The Molecular Probes® Handbook
A GUIDE TO FLUORESCENT PROBES AND LABELING TECHNOLOGIES
11th Edition (2010)
Molecular Probes™ Handbook
A Guide to Fluorescent Probes and Labeling Technologies
11th Edition (2010)
CHAPTER 1
Fluorophores
CHAPTER
11
and
Their for
Amine-Reactive
Probes
Cytoskeletal
Derivatives
Proteins
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ELEVEN
CHAPTER 11
Probes for Cytoskeletal Proteins
11.1 Probes for Actin
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
Fluorescent Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
Alexa Fluor® Actin and Unlabeled Actin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
GFP- and RFP-Labeled Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
CellLight® Null Control Reagent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480
Phallotoxins for Labeling F-Actin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480
Properties of Phallotoxin Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482
Alexa Fluor® Phalloidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482
Oregon Green® Phalloidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483
BODIPY® Phallotoxins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
Rhodamine Phalloidin and Other Red-Fluorescent Phalloidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
Other Labeled Phallotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
DNase I Conjugates for Staining G-Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
Probes for Actin Quantitation, Actin Polymerization and Actin-Binding Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485
Assays for Quantitating F-Actin and G-Actin Polymerization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485
Jasplakinolide: A Cell-Permeant F-Actin Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486
Latrunculin A and Latrunculin B: Cell-Permeant Actin Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486
Assays for Actin-Binding Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486
Data Table 11.1 Probes for Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487
Product List 11.1 Probes for Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488
11.2 Probes for Tubulin and Other Cytoskeletal Proteins
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
Paclitaxel Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
Paclitaxel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
TubulinTracker™ Green Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
Fluorescent Paclitaxel Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
Tubulin-Selective Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490
GFP- and RFP-Labeled Tubulin and MAP4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490
Anti–α-Tubulin Monoclonal Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
BODIPY® FL Vinblastine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
Other Probes for Tubulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
Probes for Other Cytoskeletal Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
GFP- and RFP-Labeled Talin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
Anti–Glial Fibrillary Acidic Protein (GFAP) Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
Anti-Desmin Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
Anti-Synapsin I Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
Data Table 11.2 Probes for Tubulin and Other Cytoskeletal Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
Product List 11.2 Probes for Tubulin and Other Cytoskeletal Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
™
The
Probes
Handbook:
A Guide
to Fluorescent
Probes
andand
Labeling
Technologies
TheMolecular
Molecular
Probes®
Handbook:
A Guide
to Fluorescent
Probes
Labeling
Technologies
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
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Chapter 11 — Probes for Cytoskeletal Proteins
Rhodamine Red™ goat anti–rabbit IgG, Alexa Fluor® 488 goat anti–mouse IgG and Hoechst 33258.
The
Molecular
Guide to
to Fluorescent
Fluorescent Probes
The
MolecularProbes®
Probes Handbook:
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Probesand
andLabeling
LabelingTechnologies
Technologies
™
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IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
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Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.1 Probes for Actin
11.1 Probes for Actin
The cytoskeleton is an essential component of a cell’s structure and
one of the easiest to label with fluorescent reagents. This section describes Molecular Probes® labeling reagents for both monomeric actin
(G-actin) and filamentous actin (F-actin); reagents for staining tubulin
and other cytoskeletal proteins are described in Section 11.2.
Fluorescent Actin
Alexa Fluor® Actin and Unlabeled Actin
Fluorescently labeled actin (Figure 11.1.1) is an important tool for investigating the structural dynamics of the cytoskeleton.1–3 We offer highly
purified actin from rabbit muscle (A12375), as well as fluorescent actin conjugates labeled with four of our brightest and most photostable dyes. The
green-fluorescent Alexa Fluor® 488 actin conjugate (A12373) has excitation
and emission maxima similar to fluorescein actin, but it is brighter and
more photostable, and its spectra are much less pH dependent. The redorange–fluorescent Alexa Fluor® 568 (A12374, Figure 11.1.2), red-fluorescent Alexa Fluor® 594 (A34050) and far-red–fluorescent Alexa Fluor® 647
(A34051) actin conjugates are more fluorescent than the spectrally similar
Lissamine rhodamine B, Texas Red® and Cy®5 conjugates, respectively.
Our fluorescent actin conjugates are prepared by reacting amine
residues of polymerized F-actin with the succinimidyl ester of the appropriate dye using a modification of the method described by Alberts
and co-workers.4 After labeling, the conjugates are subjected to depolymerization and subsequent polymerization to help ensure that the
actin conjugates are able to assemble properly. The labeled actin that
polymerizes is then separated from remaining monomeric actin by centrifugation, depolymerized and packaged in monomeric form.
Figure 11.1.1 Ribbon diagram of the structure of uncomplexed actin in the ADP state. The four
subdomains are represented in different colors, and ADP is bound at the center where the four subdomains meet. Four Ca2+ ions bound to the actin monomer are represented as gold spheres. In this
structure, tetramethylrhodamine-5-maleimide (T6027) has been used to covalently attach the dye
to a specific cysteine residue (Cys 374). Image provided by Roberto Dominguez, Boston Biomedical
Research Institute, Watertown, Massachusetts. Reprinted with permission from Science (2001)
293:708. Copyright 2001 American Association for the Advancement of Science.
GFP- and RFP-Labeled Actin
The requirement for intracellular delivery of Alexa Fluor® dye–
labeled actin conjugates by microinjection typically limits their applications for live-cell imaging to experiments involving no more than a few
(<10) cells. For applications such as high-content screening (HCS) assays
requiring larger sample sizes, GFP–actin fusions are well-established
probes for imaging cytoskeletal structure and dynamics.5 CellLight®
Actin-GFP (C10582) and CellLight® Actin-RFP (C10583, Figure 11.1.3)
Figure 11.1.3 HeLa cell labeled with CellLight® Actin-RFP (C10583) and CellLight® MAP4-GFP
(C10598) reagents and with Hoechst 33342 nucleic acid stain.
Figure 11.1.2 Chick embryo fibroblasts injected with the Alexa Fluor® 568 conjugate of actin
from rabbit muscle (A12374). The cells were then fixed and permeabilized, and the filamentous actin was stained with coumarin phallacidin (C606). The double-exposure image was
acquired using longpass filter sets appropriate for rhodamine and DAPI. Image contributed
by Heiti Paves, Laboratory of Molecular Genetics, National Institute of Chemical Physics and
Biophysics, Estonia.
™
The
Probes
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
TheMolecular
Molecular
Probes®
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
IMPORTANT
NOTICE:described
The products
described
thiscovered
manual are
covered
by one
or moreUse
Limited
Label License(s).
the Appendix
IMPORTANT NOTICE
: The products
in this
manualinare
by one
or more
Limited
LabelUse
License(s).
PleasePlease
refer refer
to thetoAppendix
on on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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479
Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.1 Probes for Actin
expression vectors (Table 11.1) generate autofluorescent proteins fused to
the N-terminus of human β-actin and incorporate all the generic advantages of BacMam 2.0 delivery technology (BacMam Gene Delivery and
Expression Technology—Note 11.1). In particular, the viral dose can be
readily adjusted to modulate expression levels if GFP- or RFP-dependent
perturbation of cellular structural or functional properties is a concern.
CellLight® Null Control Reagent
The CellLight® Null (control) reagent (C10615), a suspension of baculovirus particles lacking mammalian genetic elements, is designed for
use in parallel with our CellLight® reagents (Table 11.1). For example,
microarray expression analysis on cells treated with the CellLight® Null
(control) reagent can be used to assess down-regulation or up-regulation of host cell genes elicited by baculovirus infection.
Phallotoxins for Labeling F-Actin
We prepare a number of fluorescent and biotinylated derivatives of
phalloidin and phallacidin for selectively labeling F-actin. Phallotoxins
are bicyclic peptides isolated from the deadly Amanita phalloides mushroom 6 (www.grzyby.pl/gatunki/Amanita_phalloides.htm). They can be
used interchangeably in most applications and bind competitively to the
same sites on F-actin. Table 11.2 lists the available phallotoxin derivatives, along with their spectral properties.
A detailed staining protocol is included with each phallotoxin
derivative. One vial of the fluorescent phallotoxin contains sufficient
reagent for staining ~300 microscope slide preparations; one vial of
biotin-XX phalloidin, which must be used at a higher concentration,
contains sufficient reagent for ~50 microscope slide preparations. We
also offer unlabeled phalloidin (P3457) for blocking F-actin staining by
labeled phallotoxins and for promoting actin polymerization.
Table 11.1 CellLight® reagents and their targeting sequences.
Targeting Sequence
RFP
Handbook
GFP
Ref (489/508 nm)* (555/584 nm)* Section
Actin
Human actin
1
C10582
C10583
11.1
Tubulin
Human tubulin
2
C10613
C10614
11.2
MAP4
MAP4
3
C10598
C10599
11.2
Cat. No.
Talin
Human talin 2341–2541
4
C10611
C10612
11.2
F-Actin–Selective Probes
Chromatin
Histone 2B (H2B)
5
C10594
C10595
12.5
A22281
Mitochondria
Leader sequence
of E1α pyruvate
dehydrogenase
6
C10600
C10601
12.2
C606
Lysosomes
Lamp1 (lysosomalassociated membrane
protein 1)
7
C10596
Peroxisomes
Peroxisomal C-terminal
targeting sequence
8
C10604
Rab5a
9
C10586
C10587
12.3, 16.1
Synaptosomes Synaptophysin
10
C10609
C10610
16.1
Endoplasmic
reticulum
11
C10590
C10591
12.4
Target
Endosomes
ER signal sequence of
calreticulin and KDEL
(ER retention signal)
12.3
12.3
Human golgiresident enzyme
N-acetylgalactosaminyltransferase 2
12
Nucleus
LSV40 nuclear
localization sequence
13
C10602
C10603
12.5
Plasma
membrane
Myristoylation/
palmitoylation
sequence from Lck
tyrosine kinase
14
C10607 †
C10608 †
14.4
Golgi
apparatus
Cytoplasm
No targeting sequence
C10592
C10597
B10383
C10593
12.4
14.7
* Approximate absorption (Abs) and fluorescence (Em) maxima, in nm; GFP (Green
Fluorescent Protein) and RFP (Red Fluorescent Protein, Nat Methods (2007) 4:555) can
be imaged using optical filters for fluorescein (FITC) and tetramethylrhodamine (TRITC)
dyes, respectively. † Also available is CellLight® Plasma Membrane-CFP (C10606), which
generates a cyan-autofluorescent protein fused to the plasma membrane targeting
sequence from Lck tyrosine kinase.
1. Curr Biol (1997) 7:176; 2. PLoS One (2009) 4:e8171; 3. J Cell Biol (1995) 130:639; 4. Plant
J (2003) 33:775; 5. Curr Biol (1998) 8:377; 6. J Biol Chem (2004) 279:13044; 7. J Cell Sci (2005)
118:5243; 8. J Cell Biol (1989) 108:1657; 9. J Biol Chem (2009) 284:29218; 10. J Neurosci (2006)
26:3604; 11. FEBS Lett (1997) 405:18; 12. J Cell Biol (1998) 143:1505; 13. Trends Biochem Sci
(1991) 16:478; 14. EMBO J (1997) 16:4983.
Table 11.2 Spectral characteristics of Molecular Probes® actin-selective probes.
Actin-Selective Probe
Ex/Em *
Approximate MW
Alexa Fluor® 350 phalloidin
346/446 †
1100
Coumarin phallacidin
355/443
1100
N354
NBD phallacidin
465/536
1040
A12379
Alexa Fluor® 488 phalloidin
495/517 †
1320
F432
Fluorescein phalloidin
496/516 †
1175
O7466
Oregon Green® 488 phalloidin
496/520 †
1180
B607
BODIPY® FL phallacidin
505/512
1125
O7465
Oregon Green® 514 phalloidin
511/528 †
1280
A22282
Alexa Fluor® 532 phalloidin
528/555 †
1350
R415
Rhodamine phalloidin
540/565 †
1250
A22283
Alexa Fluor® 546 phalloidin
554/570 †
1800
A34055
Alexa Fluor® 555 phalloidin
555/565 †
1800
B3475
BODIPY® 558/568 phalloidin
558/569
1115
A12380
Alexa Fluor® 568 phalloidin
578/600 †
1590
A12381
Alexa Fluor® 594 phalloidin
593/617 †
1620
T7471
Texas Red®-X phalloidin
591/608 †
1490
A22284
Alexa Fluor® 633 phalloidin
625/645 †
1900
A34054
Alexa Fluor® 635 phalloidin
633/648 †
1900
B12382
BODIPY® 650/665 phalloidin
647/661
1200
A22287
Alexa Fluor® 647 phalloidin
649/666 †
1950
A22285
Alexa Fluor® 660 phalloidin
661/689 †
1750
A22286
Alexa Fluor® 680 phalloidin
677/699 †
1850
B7474
Biotin-XX phalloidin
NA
1300
P3457
Phalloidin
NA
790
G-Actin–Selective Probes
D12371
Alexa Fluor® 488 DNase I
495/519
>31,000
D12372
Alexa Fluor® 594 DNase I
590/617
>31,000
* Excitation (Ex) and emission (Em) maxima, in nm. Spectra of phallotoxins are either in
aqueous buffer, pH 7–9 (denoted †) or in methanol. Spectra of DNase I conjugates are in
aqueous buffer, pH 7–8. NA = Not applicable.
The
MolecularProbes®
Probes Handbook:
Handbook: A
Probesand
andLabeling
LabelingTechnologies
Technologies
The
Molecular
A Guide
Guide to
to Fluorescent
Fluorescent Probes
™
480
IMPORTANT
NOTICE:
The products
described
in this manual
aremanual
coveredare
by one
or more
Use Label
License(s).
Please
refer to thePlease
Appendix
on to
IMPORTANT
NOTICE
: The products
described
in this
covered
by Limited
one or more
Limited
Use Label
License(s).
refer
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
www.invitrogen.com/probes
thermofisher.com/probes
Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.1 Probes for Actin
NOTE 11.1
BacMam Gene Delivery and Expression Technology
Baculovirus-Mediated Transduction of Mammalian Cells
BacMam technology uses a modified insect cell baculovirus as a
vehicle to efficiently deliver and express genes in mammalian cells with
minimum effort
ffort and toxicity.1–4 We have combined the BacMam gene deff
livery and expression system with genetically encoded Premo™ sensors as
well as with genetically encoded CellLight® targeted fluorescent proteins
to yield robust and easy-to-use cell-based assays (Figure 1).
BacMam particles carrying the biosensor or targeted fluorescent
protein cDNA under the control of the CMV promoter are taken up by
endocytosis. The viral DNA traffics to the nucleus where only the CMV
promoter–driven gene is transcribed; baculovirus promoters are not
recognized by the mammalian transcriptional machinery. Following transcription, the biosensor or targeted fluorescent protein mRNA is expressed
in the cytosol and cells are soon ready to assay. This process begins within
4–6 hours after transduction and in many cell types is completed after an
overnight period.
BacMam 2.0 vectors incorporated in our CellLight® reagents extend
the applicability of BacMam-mediated transgene delivery and expression. Cells such as primary neurons that were not amenable to BacMam
transduction with version 1.0 (used in the corresponding Organelle Lights™
and Cellular Lights™ reagents) can now be transduced quantitatively in a
simple, one-step process. The improved performance is due to inclusion of
a pseudotyped capsid protein for more efficient cell entry as well as genetic
elements (enhanced CMV promoter and Woodchuck Post-Transcriptional
Regulatory Element) that boost expression levels.
Inducible, division-arrested or transient expression systems such
as the BacMam system are increasingly methods of choice to decrease
variability of expression in cell-based assays. Constitutively expressed ion
channels and other cell-surface proteins have been shown to contribute to
cell toxicity in some systems, and they may also be subject to clonal drift
and other inconsistencies that hamper successful experimentation and
screening. Moreover, the BacMam gene delivery and expression system
provides a method for simultaneously delivering multiple genes per cell,
an important feature when expressing multisubunit proteins.1
technology has many advantages when compared with lipids and other
viral delivery methods:
•
•
•
•
•
High transduction efficiency across a broad range of cell types, including primary and stem cells
Minimal microscopically observable cytopathic effects
ff
ffects
Highly reproducible and titratable transient expression
Biosafety level 1 rating (baculovirus is not pathogenic to
vertebrates and does not replicate in mammalian cells)
Ability to simultaneously deliver multiple genes
Furthermore, it is possible to divide the BacMam-transduced, homogeneous cell population into aliquots that can be stored frozen for use at a
later time, approximating the consistency of a stable cell line in a transient
expression format. More information is available at www.invitrogen.com/
handbook/bacmam2.0.
1. Nat Biotechnol (2004) 22:1583; 2. Br J Pharmacol (2008) 153:544; 3. Drug Discov
Today (2007) 12:396; 4. Nat Biotechnol (2005) 23:567; 5. Adv Virus Res (2006) 68:255.
Promoter
YFP (Venus)
Premo™ Halide Sensor gene
Baculovirus
mRNA
translated
YFP (Venus)
Endocytotic entry
mRNA
Advantages of the BacMam Delivery
and Expression System
DNA
Baculoviruses have been used extensively for protein production in
insect cells for over two decades; however, its use with mammalian cells
is relatively new. BacMam technology has opened up new avenues for
mammalian cell–based assays in drug discovery applications.3,5 In addition
to producing ready-to-use viral stocks, BacMam delivery and expression
DNA moves
to nucleus
Venus gene
transcribed
Figure 1 Schematic representation of BacMam transgene delivery and expression as exemplified by Premo™ Halide Sensor (P10229).
™
The
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
TheMolecular
MolecularProbes
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Handbook:
A Guide
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Probes
and
Labeling
Technologies
IMPORTA
T NT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
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481
Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.1 Probes for Actin
Properties of Phallotoxin Derivatives
Figure 11.1.4 Microtubules of fixed bovine pulmonary
artery endothelial cells localized with mouse monoclonal anti–α-tubulin antibody (A11126), which was subsequently visualized with Alexa Fluor® 350 goat anti–mouse
IgG antibody (A11045). Next, the F-actin was labeled with
Alexa Fluor® 594 phalloidin (A12381). Finally, the cells were
incubated with Alexa Fluor® 488 wheat germ agglutinin
(W11261) to stain components of endosomal pathways. The
superimposed and pseudocolored images were acquired
sequentially using bandpass filter sets appropriate for DAPI,
the Texas Red® dye and fluorescein, respectively.
The fluorescent and biotinylated phallotoxin derivatives stain F-actin selectively at nanomolar concentrations and are readily water soluble, thus providing convenient labels for identifying and quantitating actin in tissue sections, cell cultures or cell-free preparations.7–11
F-actin in live neurons can be efficiently labeled using cationic liposomes containing fluorescent phallotoxins, such as BODIPY® FL phallacidin 12 (B607). This procedure permits the labeling of entire cell cultures with minimum disruption. Because fluorescent phalloidin conjugates
are not permeant to most live cells, they can be used to detect cells that have compromised
membranes. However, it has been reported that unlabeled phalloidin, and potentially dyelabeled phalloidins, can penetrate the membranes of certain hypoxic cells.13 An extensive study
on visualizing the actin cytoskeleton with various fluorescent probes in cell preparations, as
well as in live cells, has been published.7
Labeled phallotoxins have similar affinity for both large and small filaments and bind in a
stoichiometric ratio of about one phallotoxin per actin subunit in both muscle and nonmuscle
cells; they reportedly do not bind to monomeric G-actin, unlike some antibodies against actin.9,14
Phallotoxins have further advantages over antibodies for actin labeling, in that 1) their binding
properties do not change appreciably with actin from different species, including plants and
animals; and 2) their nonspecific staining is negligible; thus, the contrast between stained and
unstained areas is high.
Phallotoxins shift actin’s monomer/polymer equilibrium toward the polymer, lowering
the critical concentration for polymerization as much as 30-fold.15,16 Furthermore, depolymerization of F-actin by cytochalasins, potassium iodide and elevated temperatures is
inhibited by phallotoxin binding. Because the phallotoxin derivatives are relatively small,
with approximate diameters of 12–15 Å and molecular weights below 2000 daltons, a wide
variety of actin-binding proteins—including myosin, tropomyosin, troponin and DNase I—
can still bind to actin after treatment with f luorescent phallotoxins. Even more significantly,
phallotoxin-labeled actin filaments retain certain functional characteristics; labeled glycerinated muscle fibers still contract, and labeled actin filaments still move on solid-phase
myosin substrates.17–19
Alexa Fluor® Phalloidins
We have taken advantage of the outstanding fluorescence characteristics of our Alexa Fluor®
dyes (Section 1.3) to create a series of Alexa Fluor® dye–labeled phalloidins (Figure 11.1.4, Figure
11.1.5, Figure 11.1.6, Figure 11.1.7), which are widely used F-actin stains for many applications
Figure 11.1.5 Actin filaments of the turbellarian flatworm
Archimonotresis sp. stained with Alexa Fluor® 488 phalloidin
(A12379) to reveal a meshwork of longitudinal, circular and
diagonal muscles. The large, bright ring with muscle fibers
radiating outward is the muscular pharynx, and the small,
bright ring at the posterior is part of the reproductive system. This epifluorescence image was contributed by Matthew
D. Hooge and Seth Tyler, University of Maine, Orono.
Figure 11.1.6 Subcellular structures in fixed and permeabilized bovine pulmonary artery endothelial cells visualized
with several fluorescent dyes. Filamentous actin (F-actin) was
identified with Alexa Fluor® 633 phalloidin (A22284), which
is pseudocolored magenta. Intracellular membranes were
stained with green-fluorescent DiOC6(3) (D273). Finally, nuclei were counterstained with blue-fluorescent DAPI (D1306,
D3571, D21490). The image was acquired using filters appropriate for fluorescein and DAPI and a special filter (courtesy of
Omega® Optical) for the Alexa Fluor® 633 dye, consisting of a
narrow band exciter (630DF10), dichroic (640DRLP) and emitter (660DF10).
Figure 11.1.7 FluoCells® prepared slide #4 (F24631) contains a section of mouse intestine stained with a combination of fluorescent stains. Alexa Fluor® 350 wheat germ agglutinin (W11263) is a blue-fluorescent lectin that was used
to stain the mucus of goblet cells. The filamentous actin
prevalent in the brush border was stained with red-orange–
fluorescent Alexa Fluor® 568 phalloidin (A12380). Finally, the
nuclei were stained with SYTOX® Green nucleic acid stain
(S7020). This image is a composite of three digitized images
obtained with filter sets appropriate for fluorescein, DAPI
and tetramethylrhodamine.
TheMolecular
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Probes Handbook:
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and Labeling
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GuidetotoFluorescent
Fluorescent Probes
Probes and
™
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or more
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the Appendix on
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Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.1 Probes for Actin
across the full spectral range. The Alexa Fluor® phalloidin conjugates
(Figure 11.1.8) provide researchers with fluorescent probes that are
superior in brightness and photostability to other spectrally similar
conjugates tested (Figure 11.1.9). For improved fluorescence detection
of F-actin in fixed and permeabilized cells, we encourage researchers
to try these fluorescent phalloidins in their actin-labeling protocols. A
series of videos showing Alexa Fluor® 488 phalloidin–stained actin 20
is available at the Journal of Cell Biology web site (www.jcb.org/cgi/
content/full/150/2/361/DC1).
rapidly, making their photography difficult. We have used two of our
Oregon Green® dyes (Section 1.5) to prepare Oregon Green® 488 phalloidin (O7466, Figure 11.1.10) and the slightly longer-wavelength Oregon
Green® 514 phalloidin (O7465). The excitation and emission spectra of
the Oregon Green® 488 dye are virtually superimposable on those of fluorescein, and both the Oregon Green® 488 and Oregon Green® 514 dyes
may be viewed with standard fluorescein optical filter sets. As shown in
Figure 11.1.11, Oregon Green® 514 phalloidin is more photostable than
fluorescein phalloidin, making it easier to visualize and photograph.
Oregon Green® Phalloidins
Green-fluorescent actin stains are popular reagents for labeling
F-actin in fixed and permeabilized cells. Unfortunately, the greenfluorescent fluorescein phalloidin and NBD phallacidin photobleach
SO3
H2N
O
SO3
NH2
C O
OH
O
CH3CH
C
NH
O C
HO
NH
O
O
6
CH3CCH2NH C
CH2
CH C NH CH C O
H2C
O
NH
S
CHCH3
H2C
N
H
N C CH NH C CH NH C O
O
5
O CHCH3
OH
Figure 11.1.8 Alexa Fluor® 488 phalloidin (A12379).
Figure 11.1.10 Simultaneous visualization of F- and G-actin in a bovine pulmonary artery endothelial cell (BPAEC) using F-actin–specific Oregon Green® 488 phalloidin (O7466) and G-actin–
specific Texas Red® deoxyribonuclease I. The G-actin appears as diffuse red fluorescence that
is more intense in the nuclear region where the cell thickness is greater and stress fibers are less
dense. The image was obtained by taking multiple exposures through bandpass optical filter sets
appropriate for fluorescein and the Texas Red® dye.
Fluorescence (% of initial)
100
80
60
Oregon Green® 514
40
20
Fluorescein
0
0
20
40
60
80
100
Time (seconds)
Figure 11.1.9 Comparison of the photobleaching rates of the Alexa Fluor® 488 and Alexa Fluor® 546 dyes and the well-known
fluorescein and Cy®3 fluorophores. The cytoskeleton of bovine pulmonary artery endothelial cells (BPAEC) was labeled with
(top series) Alexa Fluor® 488 phalloidin (A12379) and mouse monoclonal anti–α-tubulin antibody (A11126) in combination
with Alexa Fluor® 546 goat anti–mouse IgG antibody (A11003) or (bottom series) fluorescein phalloidin (F432) and the anti–αtubulin antibody in combination with a commercially available Cy®3 goat anti–mouse IgG antibody. The pseudocolored images were taken at 30-second intervals (0, 30, 90 and 210 seconds of exposure). The images were acquired with bandpass filter
sets appropriate for fluorescein and rhodamine.
Figure 11.1.11 Photostability comparison for Oregon
Green® 514 phalloidin (O7465) and fluorescein phalloidin
(F432). CRE BAG 2 fibroblasts were fixed with formaldehyde,
permeabilized with acetone and then stained with the fluorescent phallotoxins. Samples were continuously illuminated and images were acquired every 5 seconds using a Star
1 CCD camera (Photometrics); the average fluorescence intensity in the field of view was calculated with Image-1 software (Universal Imaging Corp.) and expressed as a fraction
of the initial intensity. Three data sets, representing different
fields of view, were averaged for each labeled phalloidin to
obtain the plotted time courses.
™
The
Probes
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
TheMolecular
Molecular
Probes®
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
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NOTICE:described
The products
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or moreUse
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on on
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483
Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.1 Probes for Actin
BODIPY® Phallotoxins
Figure 11.1.12 Permeabilized bovine pulmonary artery endothelial cells stained with SYTOX® Green nucleic acid stain
(S7020) to label the nuclei and with BODIPY® TR-X phallacidin (B7464) to label the F-actin. The image was acquired by
taking sequential exposures through bandpass optical filter
sets appropriate for fluorescein and the Texas Red® dye.
BODIPY® phallotoxin conjugates (B607, B3475, B12382; Figure 11.1.12, Figure 11.1.13) have
some important advantages over the conventional NBD, fluorescein and rhodamine phallotoxins. BODIPY® dyes are more photostable than these traditional fluorophores 21 and have narrower
emission bandwidths (Section 1.4), making them especially useful for double- and triple-labeling
experiments. BODIPY® FL phallacidin (B607), which reportedly gives a signal superior to that
of fluorescein phalloidin,22 has been used for quantitating F-actin and determining its distribution in cells.23,24
The BODIPY® FL and BODIPY® 558/568 phallotoxins (B607, B3475) exhibit excitation
and emission spectra similar to those of fluorescein and rhodamine B, respectively, and can
be used with standard optical filter sets. BODIPY® 650/665 phalloidin (B12382) is the longestwavelength BODIPY® phallotoxin conjugate available, increasing the options for multicolor
analysis. BODIPY® 650/665 phalloidin, Alexa Fluor® 647 phalloidin (A22287) and Alexa Fluor®
660 phalloidin (A22285) are among the few probes available that can be excited by the 647 nm
spectral line of the Ar-Kr laser.
Rhodamine Phalloidin and Other Red-Fluorescent Phalloidins
Rhodamine phalloidin (R415, Figure 11.1.14) has been the standard for red-fluorescent phallotoxins.25–27 Rhodamine phalloidin is excited efficiently by the mercury-arc lamp in most fluorescence microscopes. However, our Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594 and
Texas Red®-X phalloidins 28 (A22283, A12380, A12381, T7471; Figure 11.1.15, Figure 11.1.16) will
be welcome replacements for rhodamine phalloidin in many multicolor applications because
their emission spectra are better separated from those of the green-fluorescent Alexa Fluor® 488,
Oregon Green® and fluorescein dyes.
Other Labeled Phallotoxins
Figure 11.1.13 Actin labeled with BODIPY® FL phallacidin
(B607) and vinculin, a cytoskeletal focal adhesion protein,
tagged with a monoclonal anti-vinculin antibody that was
subsequently probed with Texas Red® goat anti–mouse IgG antibody (T862). The large triangular cell is a fibroblast containing
green actin stress fibers terminating in red focal adhesions. The
neighboring polygonal cell, a rat neonatal cardiomyocyte, contains green striated actin in the myofibrils terminating in the focal adhesions. The close apposition of the two stains results in a
yellowish-orange color. Image contributed by Mark B. Snuggs
and W. Barry VanWinkle, University of Texas, Houston.
The original yellow-green–fluorescent NBD phallacidin (N354) and green-fluorescent
fluorescein phalloidin (F432) remain in use despite their relatively poor photostability (Figure
11.1.11). Photostability of fluorescein phalloidin and some other fluorescent phallotoxins can
be considerably improved (Figure 11.1.17) by mounting the stained samples with our ProLong®
Antifade Kit or ProLong® Gold antifade reagent (P7481, P36930, P36934; Section 23.1). We recommend the Alexa Fluor® 488, Oregon Green® 488, Oregon Green® 514 and BODIPY® FL phallotoxins for photostable, green-fluorescent actin staining. Alexa Fluor® 350 phalloidin (A22281)
and coumarin phallacidin (C606, Figure 11.1.2) are the only blue-fluorescent phallotoxin conjugates currently available for staining actin.29
Biotin-XX phalloidin (B7474) permits detection of F-actin by electron microscopy and light
microscopy techniques.30 This biotin conjugate can be visualized with fluorophore- or enzymelabeled avidin and streptavidin (Section 7.6) or with tyramide signal amplification (TSA™) technology (Section 6.2). Biotin-XX phalloidin, in conjunction with streptavidin or CaptAvidin™
agarose (S951, C21386; Section 7.6), can be used to precipitate F-actin from the cytosolic antiphosphotyrosine–reactive fraction in macrophages stimulated with colony-stimulating factor-1. 31
DNase I Conjugates for Staining G-Actin
Figure 11.1.14 Actin filaments of chick heart fibroblasts
stained with rhodamine phalloidin (R415). The subcompartments in the cytoskeleton are readily apparent and labeled
as follows: sf, stress fiber; lam, lamellipodium; fil/ms, filipodium/microspike; am, actin meshwork; arc, dorsal arc. Figure
reprinted from "Visualizing the Actin Cytoskeleton." J. Small et
al. Microscopy Research and Technique (1999) 47:3. Reprinted
by permission of Wiley-Liss, Inc., a subsidiary of John Wiley &
Sons, Inc., and J. Victor Small.
Bovine pancreatic deoxyribonuclease (DNase I, ~31,000 daltons) binds much more strongly
to monomeric G-actin than to filamentous F-actin, with binding constants of 5 × 108 M–1 and
1.2 × 104 M–1, respectively.32–35 Because of this strong, selective binding to G-actin, fluorescent
DNase I conjugates have proven very useful for detecting and quantitating the proportion of
unpolymerized actin in a cell. We have triple-labeled endothelial cells with fluorescein DNase
I, BODIPY® 581/591 phalloidin and a monoclonal anti-actin antibody detected with a Cascade
Blue® dye–labeled secondary antibody 36 (C962, Section 7.2). We found that the monoclonal antibody, which binds to both G-actin and F-actin, colocalized with the DNase I and phalloidin conjugates, suggesting that these three probes recognize unique binding sites on the actin
molecule. Researchers can choose DNase I conjugates labeled with either the green-fluorescent
Alexa Fluor® 488 (D12371) or red-fluorescent Alexa Fluor® 594 (D12372) dyes, depending on their
multicolor application and their detection instrumentation (Table 11.2).
The
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
and Labeling
LabelingTechnologies
Technologies
The
Molecular
Guideto
toFluorescent
Fluorescent Probes
Probes and
™
484
IMPORTANT
NOTICE:
The products
described
in this manual
covered
bycovered
one or more
Limited
Use Label
License(s).
Please
refer to thePlease
Appendix
IMPORTANT
NOTICE
: The products
described
in thisare
manual
are
by one
or more
Limited
Use Label
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referonto
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
www.invitrogen.com/probes
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Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.1 Probes for Actin
Alexa Fluor® 488 and Alexa Fluor® 594 DNase I conjugates have been used in combination
with fluorescently labeled phallotoxins to simultaneously visualize G-actin pools and filamentous F-actin 37,38 and to study the disruption of microfilament organization in live nonmuscle
cells.39 Rhodamine phalloidin (R415) has been used in conjunction with Oregon Green® 488
DNase I to determine the F-actin:G-actin ratio in Dictyostelium using confocal laser-scanning
microscopy.40 A mouse fibroblast labeled with both Texas Red® DNase I and Oregon Green® 488
phalloidin (O7466) permitted visualization of the G-actin and the complex network of F-actin
throughout the cytoplasm, as well as at the cell periphery (Figure 11.1.10). The influence of cytochalasins on actin structure in monocytes has been quantitated by flow cytometry using Texas
Red® DNase I and BODIPY® FL phallacidin (B607) to stain the G-actin and F-actin pools, respectively.41 Fluorescent DNase I has also been used as a model system to study the interactions
of nucleotides, cations and cytochalasin D with monomeric actin.42
Probes for Actin Quantitation, Actin
Polymerization and Actin-Binding Proteins
Assays for Quantitating F-Actin and G-Actin Polymerization
Quantitative assays for F-actin have employed fluorescein phalloidin,43,44 rhodamine phalloidin,45 BODIPY® FL phallacidin 24 and NBD phallacidin.46 An F-actin assay based on fluorescein phalloidin was used to demonstrate the loss of F-actin from cells during apoptosis.47 The addition of propidium iodide (P1304MP, P3566, P21493; Section 8.1) to the cell suspensions enabled
these researchers to estimate the cell-cycle distributions of both the apoptotic and nonapoptotic
cell populations. The change in F-actin content in proliferating adherent cells has been quantitated using the ratio of rhodamine phalloidin fluorescence to ethidium bromide fluorescence.48
The spectral separation of the signals in this assay may be improved by using a green-fluorescent
stain for F-actin and a high-affinity red-fluorescent nucleic acid stain, such as the combination of
Alexa Fluor® 488 phalloidin (A12379) and ethidium homodimer-1 (E1169, Section 8.1).
The fluorescence of actin monomers labeled with pyrene iodoacetamide (P29) has been demonstrated to change upon polymerization, making this probe an excellent tool for following the
kinetics of actin polymerization and the effects of actin-binding proteins on polymerization.49–51
Figure 11.1.15 A section of mouse intestine stained with a
combination of fluorescent stains. Fibronectin, an extracellular matrix adhesion molecule, was labeled using a chicken
primary antibody against fibronectin and visualized using
green-fluorescent Alexa Fluor® 488 goat anti–chicken IgG
antibody (A11039). The filamentous actin (F-actin) prevalent in the brush border was stained with red-fluorescent
Alexa Fluor® 568 phalloidin (A12380). Finally, the nuclei were
stained with DAPI (D1306, D3571, D21490).
Figure 11.1.16 Confocal micrograph of the cytoskeleton of a mixed population of granule neurons and glial
cells. The F-actin was stained with red-fluorescent Texas
Red®-X phalloidin (T7471). The microtubules were detected with a mouse monoclonal anti–ß-tubulin primary
antibody and subsequently visualized with the green-fluorescent Alexa Fluor® 488 goat anti–mouse IgG antibody
(A11001). The image was contributed by Jonathan Zmuda,
Immunomatrix, Inc.
Figure 11.1.17 Bovine pulmonary artery endothelial cells were labeled with fluorescein phalloidin (F432), which labels filamentous actin, and placed under constant illumination on the microscope with a FITC filter set using a 60× objective. Images
were acquired at one-second intervals for 30 seconds. Under these illumination conditions, fluorescein photobleached to
about 12% of its initial value in 30 seconds in PBS (left), but stayed at the initial value under the same illumination conditions
when mounted using the reagents in the ProLong® Antifade Kit (right, P7481).
™
The
Probes
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
TheMolecular
Molecular
Probes®
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
IMPORTANT NOTICE
: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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485
Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.1 Probes for Actin
Jasplakinolide: A Cell-Permeant F-Actin Probe
Figure 11.1.18 Jasplakinolide (J7473).
We offer jasplakinolide (J7473, Figure 11.1.18), a macrocyclic peptide isolated from the
marine sponge Jaspis johnstoni.52–54 Jasplakinolide is a potent inducer of actin polymerization
in vitro by stimulating actin filament nucleation 55,56 and competes with phalloidin for actin
binding 57 (Kd = 15 nM). Moreover, unlike other known actin stabilizers such as phalloidins and
virotoxins, jasplakinolide appears to be somewhat cell permeant and therefore can potentially
be used to manipulate actin polymerization in live cells. This peptide, which also exhibits fungicidal, insecticidal and antiproliferative activity, 53,58–60 is particularly useful for investigating
cell processes mediated by actin polymerization and depolymerization, including cell adhesion,
locomotion, endocytosis and vesicle sorting and release. Jasplakinolide has been reported to
enhance apoptosis induced by cytokine deprivation.61
Latrunculin A and Latrunculin B: Cell-Permeant Actin Antagonists
Latrunculins are powerful disruptors of microfilament organization. Isolated from a Red Sea
sponge, these G-actin binding compounds inhibit fertilization and early embryological development,62 alter the shape of cells 63,64 and inhibit receptor-mediated endocytosis.65 Latrunculin
A 61,63,66 (L12370, Figure 11.1.19) binds to monomeric G-actin in a 1:1 ratio at submicromolar concentrations (Howard Petty, Wayne State University, personal communication) and is frequently
used to establish the effects of F-actin disassembly on particular physiological functions such as
ion transport 67 and protein localization.68 The activity of latrunculin B (L22290) mimics that of
latrunculin A in most applications.63,65,69–71
Figure 11.1.19 Latrunculin A (L12370).
Assays for Actin-Binding Proteins
Enhancement of the fluorescence of certain phallotoxins upon binding to F-actin can be a
useful tool for following the kinetics and extent of binding of specific actin-binding proteins. We
have used the change in fluorescence of rhodamine phalloidin (R415) to determine the dissociation constant of various phallotoxins.72 The enhancement of rhodamine phalloidin’s fluorescence
upon actin binding has also been used to measure the kinetics and extent of gelsolin severing of
actin filaments.73 The affinity and rate constants for rhodamine phalloidin binding to actin are
not affected by saturation of actin with either myosin subfragment-1 or tropomyosin, indicating
that these two actin-binding proteins do not bind to the same sites as the phalloidin.12
REFERENCES
1. J Cell Biol (2009) 185:323; 2. J Am Chem Soc (2008) 130:16840; 3. Biophys J (2007) 92:1081; 4. Development (1988)
103:675; 5. Mol Biotechnol (2002) 21:241; 6. Proc Natl Acad Sci U S A (1974) 71:2803; 7. Microsc Res Tech (1999)
47:3; 8. Biophys J (1998) 74:2451; 9. Biophys J (2005) 88:2727; 10. Methods Enzymol (1991) 194:729; 11. J Muscle Res
Cell Motil (1988) 9:370; 12. Neurosci Lett (1996) 207:17; 13. J Lab Clin Med (1994) 123:357; 14. Biochemistry (1994)
33:14387; 15. Eur J Biochem (1987) 165:125; 16. J Cell Biol (1987) 105:1473; 17. J Cell Biol (1991) 115:67; 18. Nature
(1987) 326:805; 19. Proc Natl Acad Sci U S A (1986) 83:6272; 20. J Cell Biol (2000) 150:361; 21. J Cell Biol (1991)
114:1179; 22. J Cell Biol (1994) 127:1637; 23. J Cell Biol (1992) 116:197; 24. Histochem J (1990) 22:624; 25. Biochemistry
(2008) 47:6460; 26. BMC Cell Biol (2007) 8:43; 27. Biotechniques (2006) 40:745; 28. J Histochem Cytochem (2001)
49:1351; 29. J Muscle Res Cell Motil (1993) 14:594; 30. J Cell Biol (1995) 130:591; 31. Biol Chem (1998) 273:17128;
32. Anal Biochem (1983) 135:22; 33. Exp Cell Res (1983) 147:240; 34. Eur J Biochem (1980) 104:367; 35. J Biol Chem
(1980) 255:5668; 36. J Histochem Cytochem (1994) 42:345; 37. Stem Cells (2005) 23:507; 38. Am J Physiol Heart Circ
Physiol (2005) 288:H660; 39. Proc Natl Acad Sci U S A (1990) 87:5474; 40. J Cell Biol (1998) 142:1325; 41. J Biol Chem
(1994) 269:3159; 42. Eur J Biochem (1989) 182:267; 43. Proc Natl Acad Sci U S A (1980) 77:6624; 44. J Cell Sci (1991)
100:187; 45. J Cell Biol (1995) 130:613; 46. J Cell Biol (1984) 98:1265; 47. Cytometry (1995) 20:162; 48. J Cell Biol
(1995) 129:1589; 49. Curr Biol (2006) 16:1924; 50. J Biol Chem (2008) 283:7135; 51. Biophys J (2007) 92:2162; 52. J Cell
Biol (1997) 137:399; 53. J Am Chem Soc (1986) 108:3123; 54. Tetrahedron Lett (1986) 27:2797; 55. Methods Mol Biol
(2001) 161:109; 56. J Biol Chem (2000) 275:5163; 57. J Biol Chem (1994) 269:14869; 58. J Natl Cancer Inst (1995) 87:46;
59. Cancer Chemother Pharmacol (1992) 30:401; 60. Antimicrob Agents Chemother (1988) 32:1154; 61. J Biol Chem
(1999) 274:4259; 62. Science (1983) 219:493; 63. J Biol Chem (2000) 275:28120; 64. FEBS Lett (1987) 213:316; 65. Exp
Cell Res (1986) 166:191; 66. Cell Motil Cytoskeleton (1989) 13:127; 67. J Biol Chem (1997) 272:20332; 68. Am J Physiol
(1997) 272:C254; 69. J Biol Chem (2001) 276:23056; 70. J Cell Sci (2001) 114:1025; 71. Cell Motil Cytoskeleton (2001)
48:96; 72. Anal Biochem (1992) 200:199; 73. J Biol Chem (1994) 269:32916.
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Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.1 Probes for Actin
DATA TABLE 11.1 PROBES FOR ACTIN
Cat. No.
MW
Storage
Soluble
Abs
EC
Em
Solvent
Notes
A12379
~1320
F,L
MeOH, H2O
494
78,000
517
pH 7
1, 2, 3
578
88,000
600
pH 7
1, 2, 3
A12380
~1590
F,L
MeOH, H2O
593
92,000
617
pH 7
1, 2, 3
A12381
~1620
F,L
MeOH, H2O
346
17,000
446
pH 7
1, 2, 3
A22281
~1100
F,L
MeOH, H2O
528
81,000
555
pH 7
1, 2, 3
A22282
~1350
F,L
MeOH, H2O
554
112,000
570
pH 7
1, 2, 3
A22283
~1800
F,L
MeOH, H2O
621
159,000
639
MeOH
1, 2, 3, 4
A22284
~1900
F,L
MeOH, H2O
668
132,000
697
MeOH
1, 2, 3, 4
A22285
~1650
F,L
MeOH, H2O
684
183,000
707
MeOH
1, 2, 3, 4
A22286
~1850
F,L
MeOH, H2O
650
275,000
672
MeOH
1, 2, 3, 4
A22287
~1950
F,L
MeOH, H2O
622
145,000
640
MeOH
1, 2, 3, 4
A34054
~1800
F,L
MeOH, H2O
555
155,000
572
MeOH
1, 2, 3
A34055
~1900
F,L
MeOH, H2O
505
83,000
512
MeOH
1, 2, 3
B607
~1160
F,L
MeOH, H2O
558
85,000
569
MeOH
1, 2, 3
B3475
~1115
F,L
MeOH, H2O
<300
none
1, 2
B7474
~1300
F
MeOH, H2O
B12382
~1200
F,L
MeOH
647
102,000
661
MeOH
1, 3, 5
355
16,000
443
MeOH
1, 2, 3
C606
~1100
F,L
MeOH, H2O
496
84,000
516
pH 8
1, 2, 3
F432
~1175
F,L
MeOH, H2O
J7473
709.68
F,D
MeOH
278
8000
none
MeOH
L12370
421.55
F,D
DMSO
<300
none
L22290
395.51
F,D
DMSO
<300
none
465
24,000
536
MeOH
1, 2, 3
N354
~1040
F,L
MeOH, H2O
511
85,000
528
pH 9
1, 2, 3
O7465
~1280
F,L
MeOH, H2O
496
86,000
520
pH 9
1, 2, 3
O7466
~1180
F,L
MeOH, H2O
P29
385.20
F,D,L
DMF, DMSO
339
26,000
384
MeOH
6, 7
<300
see Notes
2, 8
P3457
~790
F
MeOH, H2O
542
85,000
565
MeOH
1, 2, 3, 9
R415
~1250
F,L
MeOH, H2O
583
95,000
603
MeOH
1, 2, 3, 9
T7471
~1490
F,L
MeOH, H2O
For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.
Notes
1. α-Bungarotoxin, EGF and phallotoxin conjugates have approximately 1 label per peptide.
2. Although this phallotoxin is water-soluble, storage in water is not recommended, particularly in dilute solution.
3. The value of EC listed for this phallotoxin conjugate is for the labeling dye in free solution. Use of this value for the conjugate assumes a 1:1 dye:peptide labeling ratio and no change of EC due to
dye–peptide interactions.
4. In aqueous solutions (pH 7.0), Abs/Em = 625/645 nm for A22284, 633/648 nm for A34054, 649/666 nm for A22287, 661/689 nm for A22285 and 677/699 nm for A22286.
5. B7464 and B12382 are not directly soluble in H2O. Aqueous dispersions can be prepared by dilution of a stock solution in MeOH.
6. Spectral data of the 2-mercaptoethanol adduct.
7. Iodoacetamides in solution undergo rapid photodecomposition to unreactive products. Minimize exposure to light prior to reaction.
8. This bicyclic peptide is very weakly fluorescent in aqueous solution (Em ~380 nm). (Biochim Biophys Acta (1983) 760:411)
9. In aqueous solutions (pH 7.0), Abs/Em = 554/573 nm for R415 and 591/608 nm for T7471.
™
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487
Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.1 Probes for Actin
PRODUCT LIST 11.1 PROBES FOR ACTIN
Cat. No.
Product
A12375
A12373
A12374
A34050
A34051
A22281
A12379
A22282
A22283
A34055
A12380
A12381
A22284
A34054
A22287
A22285
A22286
B7474
B3475
B12382
B607
C10582
C10583
C10615
C606
D12371
D12372
F432
J7473
L12370
L22290
N354
O7466
O7465
P3457
P29
R415
T7471
actin from rabbit muscle
actin from rabbit muscle, Alexa Fluor® 488 conjugate *in solution*
actin from rabbit muscle, Alexa Fluor® 568 conjugate *in solution*
actin from rabbit muscle, Alexa Fluor® 594 conjugate *in solution*
actin from rabbit muscle, Alexa Fluor® 647 conjugate *in solution*
Alexa Fluor® 350 phalloidin
Alexa Fluor® 488 phalloidin
Alexa Fluor® 532 phalloidin
Alexa Fluor® 546 phalloidin
Alexa Fluor® 555 phalloidin
Alexa Fluor® 568 phalloidin
Alexa Fluor® 594 phalloidin
Alexa Fluor® 633 phalloidin
Alexa Fluor® 635 phalloidin
Alexa Fluor® 647 phalloidin
Alexa Fluor® 660 phalloidin
Alexa Fluor® 680 phalloidin
biotin-XX phalloidin
BODIPY® 558/568 phalloidin
BODIPY® 650/665 phalloidin
BODIPY® FL phallacidin
CellLight® Actin-GFP *BacMam 2.0*
CellLight® Actin-RFP *BacMam 2.0*
CellLight® Null (control) *BacMam 2.0*
coumarin phallacidin
deoxyribonuclease I, Alexa Fluor® 488 conjugate
deoxyribonuclease I, Alexa Fluor® 594 conjugate
fluorescein phalloidin
jasplakinolide
latrunculin A
latrunculin B
N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin (NBD phallacidin)
Oregon Green® 488 phalloidin
Oregon Green® 514 phalloidin
phalloidin
N-(1-pyrene)iodoacetamide
rhodamine phalloidin
Texas Red®-X phalloidin
Quantity
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1 mg
200 µg
200 µg
200 µg
200 µg
300 U
300 U
300 U
300 U
300 U
300 U
300 U
300 U
300 U
300 U
300 U
300 U
50 U
300 U
300 U
300 U
1 mL
1 mL
1 mL
300 U
5 mg
5 mg
300 U
100 µg
100 µg
100 µg
300 U
300 U
300 U
1 mg
100 mg
300 U
300 U
Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.2 Probes for Tubulin and Other Cytoskeletal Proteins
11.2 Probes for Tubulin and Other Cytoskeletal Proteins
Paclitaxel Probes
Paclitaxel
We offer paclitaxel (P3456) for research purposes only at a purity
of >98% by HPLC. Paclitaxel, formerly referred to as taxol in some
scientific literature, is the approved generic name for the anticancer
pharmaceutical Taxol® (Bristol-Myers Squibb Co.). The diterpenoid
paclitaxel is a potent anti-neoplastic agent 1,2 originally isolated from
the bark and needles of the western yew tree, Taxus brevifolia. 3,4 The
anti-mitotic and cytotoxic action of paclitaxel is related to its ability
to promote tubulin assembly into stable aggregated structures that
cannot be depolymerized by dilution, calcium ions, cold or a number of microtubule-disrupting drugs; 5–7 paclitaxel also decreases the
critical concentration of tubulin required for microtubule assembly. Cultured cells treated with paclitaxel are blocked in the G2 (the
"gap" between DNA synthesis and mitosis) and M (mitosis) phases
of the cell cycle. 8
TubulinTracker™ Green Reagent
TubulinTracker™ Green reagent (T34075) provides green-fluorescent staining of polymerized tubulin in live cells.9–11 Also known as
Oregon Green® 488 paclitaxel bis-acetate (a bi-acetylated version of
Oregon Green® 488 paclitaxel (P22310), see below), TubulinTracker™
Green reagent is an uncharged, nonfluorescent compound (Figure
11.2.1) that easily passes through the plasma membrane of live cells. Once
inside the cell, the lipophilic blocking group is cleaved by nonspecific
esterases, resulting in a green-fluorescent, charged paclitaxel.
O
TubulinTracker™ Green reagent is provided as a set of two components: lyophilized TubulinTracker™ Green reagent and a 20% Pluronic®
F-127 solution in dimethylsulfoxide (DMSO), a solubilizing agent for
making stock solutions and facilitating cell loading. Please note that
because paclitaxel binds polymerized tubulin, TubulinTracker™ Green
reagent will inhibit cell division and possibly other functions utilizing
polymerized tubulin in live cells.
Fluorescent Paclitaxel Conjugates
In addition to unlabeled paclitaxel and TubulinTracker™ Green reagent, we provide three fluorescent derivatives of paclitaxel: Oregon
Green® 488 paclitaxel (Flutax-2, P22310), BODIPY® FL paclitaxel
(P7500) and BODIPY® 564/570 paclitaxel (P7501). These fluorescent
paclitaxel derivatives are promising tools for imaging microtubule
formation and motility. Their fluorescent attributes should also make
these conjugates useful reagents for screening compounds that affect
microtubule assembly.
Oregon Green® 488 paclitaxel 12–16 is an important probe for labeling tubulin filaments in live cells. The fluorescent label on this probe
is attached by derivatizing the 7β-hydroxy group of native paclitaxel
(Figure 11.2.2), a strategy that permits selective binding of the probe to
microtubules with high affinity at 37°C 16 (Kd ~10 –7 M). Oregon Green®
488 paclitaxel has been utilized in a high-throughput fluorescence polarization–based assay to screen for paclitaxel biomimetics.14 We have
successfully used Oregon Green® 488 paclitaxel to label microtubules
O
CH3CO
O
F
OCCH3
F
O
C O
CH3
C O
O
O
O
H3C
O
CH3
CH3
CH3
C NHCHCH C O
OH
HO
O O
O C C O
CH3
O
C O
O C CH2CH2NH
O
Figure 11.2.1 TubulinTracker™ Green (Oregon Green® 488 Taxol®, bis-acetate; T34075).
Figure 11.2.2 Paclitaxel, Oregon Green® 488 conjugate (Oregon Green® 488 Taxol®,
Flutax-2; P22310).
™
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Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.2 Probes for Tubulin and Other Cytoskeletal Proteins
of live HeLa (Figure 11.2.3), NIH 3T3, A-10 and BC3H1 cells. Xenopus
laevis17 and bovine brain 18 microtubules have also been stained with
Oregon Green® 488 paclitaxel.
In the BODIPY® FL and BODIPY® 564/570 paclitaxel derivatives,
the N-benzoyl substituent of the 3-phenylisoserine portion of native
paclitaxel is replaced by a BODIPY® propionyl substituent (Figure
11.2.4). As an alternative to chemically modifying tubulin with a reactive fluorophore, a published method describes the use of these
BODIPY® paclitaxel derivatives to generate fluorescent microtubules
that are stable at room temperature for one week or longer.19 In contrast
to the Oregon Green® 488 derivative, the BODIPY® FL and BODIPY®
564/570 paclitaxel derivatives do not appear to be suitable for labeling
intracellular tubulin in most cases.
Tubulin-Selective Probes
Figure 11.2.3 Microtubules were assembled, stabilized and visualized with the aid of greenfluorescent Oregon Green® 488 paclitaxel (P22310). Viable HeLa cells were incubated with the
conjugate for 1 hour, followed by several washes with phosphate-buffered saline containing
2% bovine serum albumin. The image was acquired using a confocal laser-scanning microscope and a filter set appropriate for fluorescein.
GFP- and RFP-Labeled Tubulin and MAP4
GFP–tubulin fusions are well-established probes for imaging cytokinesis and other dynamic rearrangements of microtubules in live
cells.20 CellLight® Tubulin-GFP and CellLight® Tubulin-RFP expression vectors (C10613, C10614; Table 11.1) generate autofluorescent proteins fused to the N-terminus of human β-tubulin and incorporate all
the generic advantages of BacMam 2.0 delivery technology (BacMam
Gene Delivery and Expression Technology—Note 11.1).
In context-specific instances where GFP–tubulin fusion protein
incorporation into microtubules is inefficient, CellLight® expression
vectors encoding GFP (C10598; Figure 11.2.5) or RFP (C10599) fused
to the N-terminus of the mammalian microtubule-associated protein
MAP4 provide a second option for microtubule visualization. However,
because MAP4 stabilizes polymerized tubulin, CellLight® Tubulin-GFP
and CellLight® Tubulin-RFP are generally preferable for molecular-level
investigations of microtubule dynamic instability.
Figure 11.2.4 Paclitaxel, BODIPY® FL conjugate (BODIPY® FL Taxol®, P7500).
Figure 11.2.5 Human mesenchymal stem cell labeled
with CellLight® MAP4-GFP (C10598) and CellLight® Histone
2B-RFP (C10595) reagents.
Figure 11.2.6 Microtubules of bovine pulmonary artery endothelial cells tagged with mouse monoclonal anti–α-tubulin antibody (A11126) and subsequently probed with: Alexa Fluor® 488 goat anti–mouse IgG antibody (A11001, left), Alexa Fluor®
546 goat anti–mouse IgG antibody (A11003, middle) or Alexa Fluor® 594 goat anti–mouse IgG antibody (A11005, right). These
images were acquired using a fluorescein bandpass optical filter set, a rhodamine bandpass optical filter set and a Texas Red®
bandpass optical filter set, respectively.
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LabelingTechnologies
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Guideto
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Fluorescent Probes
Probes and
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Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.2 Probes for Tubulin and Other Cytoskeletal Proteins
Anti–α-Tubulin Monoclonal Antibody
When used in conjunction with an anti–mouse IgG secondary immunoreagent (Section 7.2, Table 7.1), our anti–α-tubulin monoclonal
antibody (A11126) enables researchers to visualize microtubules in
fixed cells (Figure 11.2.6, Figure 11.2.7, Figure 11.2.8, Figure 11.2.9)
and in fixed or frozen tissue sections from various species. This mouse
monoclonal antibody, which recognizes amino acid residues 69–97 of
the N-terminal structural domain, is also useful for detecting tubulin
by ELISA or western blotting, for screening expression libraries and as
a probe for the N-terminal domain of α-tubulin.
The anti–α-tubulin monoclonal antibody is available either unlabeled (A11126) or as a biotin-XX conjugate (A21371). For detecting the
biotinylated antibody, we carry a wide variety of fluorophore- and enzyme-labeled avidin, streptavidin and NeutrAvidin™ biotin-binding protein conjugates and NANOGOLD® and Alexa Fluor® FluoroNanogold™
streptavidin (Section 7.6, Table 7.9).
We have extensively utilized the mouse IgG1 monoclonal anti–αtubulin antibody during development and evaluation of our Zenon®
technology (Section 7.3, Table 7.7), which allows labeling of submicrogram quantities of primary antibodies in minutes (Figure 11.2.10,
Figure 11.2.11). A comprehensive listing of our primary antibodies for cytoskeletal proteins can be found at www.invitrogen.com/
handbook/antibodies.
Figure 11.2.7 Microtubules of fixed bovine pulmonary artery endothelial cells were labeled
with our mouse monoclonal anti–α-tubulin antibody (A11126), detected with the biotin-XX–conjugated F(ab’)2 fragment of goat anti–mouse IgG antibody (B11027) and visualized with Alexa
Fluor® 488 streptavidin (S11223). The actin filaments were then labeled with orange-fluorescent
Alexa Fluor® 568 phalloidin (A12380), and the cell was counterstained with blue-fluorescent
Hoechst 33342 (H1399, H3570, H21492) to image the DNA, and red-fluorescent propidium iodide
(P1304MP, P3566, P21493) to image the nucleolar RNA. The multiple-exposure image was acquired
using bandpass filters appropriate for the Texas Red® dye, fluorescein and DAPI.
Figure 11.2.8 Bovine pulmonary artery endothelial cells were labeled with Alexa Fluor®
488 phalloidin (A12379) to stain F-actin and our mouse monoclonal anti–α-tubulin antibody
(A11126) in combination with Alexa Fluor® 594 dye–conjugated F(ab’)2 fragment of goat
anti–mouse IgG antibody (A11020) to stain microtubules. The multiple-exposure image was
acquired using bandpass filter sets appropriate for Texas Red® dye and fluorescein.
Figure 11.2.9 A zebrafish cryosection incubated with the
biotin-XX conjugate of mouse monoclonal anti–α-tubulin
antibody (A21371). The signal was amplified with TSA™ Kit
#22, which includes HRP–streptavidin and Alexa Fluor® 488
tyramide (T20932). The sample was then incubated with
the mouse monoclonal FRet 6 antibody and was visualized
with Alexa Fluor® 647 goat anti–mouse IgG (A21235), which
is pseudocolored magenta. Finally, the nuclei were counterstained with SYTOX® Orange nucleic acid stain (S11368).
Figure 11.2.10 Fixed and permeabilized bovine pulmonary artery endothelial cells stained with Alexa Fluor® 350
phalloidin (A22281), an anti–α-tubulin antibody (A11126)
and the anti–cdc6 peptide antibody (A21286). The anti–αtubulin antibody was labeled with the Zenon® Alexa Fluor®
568 Mouse IgG1 Labeling Kit (Z25006) and the anti–cdc6
peptide antibody was labeled with the Zenon® Alexa Fluor®
488 Mouse IgG1 Labeling Kit (Z25002).
Figure 11.2.11 A prometaphase muntjac skin fibroblast
stained with Alexa Fluor® 350 phalloidin (A22281), an
anti–α-tubulin antibody (A11126) and an anti–cdc6 peptide antibody (A21286). The anti–α-tubulin antibody was
prelabeled with the Zenon® Alexa Fluor® 488 Mouse IgG1
Labeling Kit (Z25002) and the anti–cdc6 peptide antibody
was prelabeled with the Zenon® Alexa Fluor® 647 Mouse
IgG1 Labeling Kit (Z25008).
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Handbook:
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Probes
and
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Technologies
TheMolecular
Molecular
Probes®
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A Guide
to Fluorescent
Probes
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Labeling
Technologies
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Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.2 Probes for Tubulin and Other Cytoskeletal Proteins
BODIPY® FL Vinblastine
BODIPY® FL vinblastine (V12390, Figure 11.2.12), a fluorescent
analog of the anticancer drug vinblastine, is a useful probe for labeling β-tubulin and for investigating drug-transport mechanisms.21,22
Vinblastine inhibits cell proliferation by capping microtubule ends,
thereby suppressing mitotic spindle microtubule dynamics.23 Another
fluorescent vinblastine derivative, vinblastine 4´-anthranilate, reportedly binds to the central portion of the primary sequence of β-tubulin
and inhibits polymerization.21,24–26
In addition, intracellular accumulation of vinblastine has been associated with a vinblastine-specific modulating site on P-glycoprotein,
a drug-efflux pump that is overexpressed in multidrug-resistant (MDR)
cells 27 (Section 15.6). This highly lipophilic P-glycoprotein substrate
has also been used to study the role of P-glycoprotein in drug penetration through the blood-brain barrier.28 Fluorescently labeled vinblastine analogs, including BODIPY® FL vinblastine, have been employed
to measure drug-transport kinetics in MDR cells.29
Other Probes for Tubulin
The nuclear stain DAPI (D1306, D3571, D21490) binds tightly to
purified tubulin in vitro without interfering with microtubule assembly
or GTP hydrolysis. DAPI binds to tubulin at sites different from those of
paclitaxel, colchicine and vinblastine, and its binding is accompanied
by shifts in the absorption spectra and fluorescence enhancement. The
affinity of DAPI for polymeric tubulin is 7-fold greater than for dimeric
tubulin, making DAPI a sensitive tool for investigating microtubule
assembly kinetics.30–33 DAPI has been used to screen for potential antimicrotubule drugs in a high-throughput assay.34
Bis-ANS (B153) is a potent inhibitor of in vitro microtubule assembly. 35 This fluorescent probe binds to the hydrophobic clefts of
proteins with an affinity approximately 10–100 times higher than that
of 1,8-ANS (A47, Section 13.5) and exhibits a significant fluorescence
enhancement upon binding. The bis-ANS binding site on tubulin lies
near the critical contact region for microtubule assembly, but it is distinct from the binding sites for colchicine, vinblastine, podophyllotoxin and maytansine. 36–38 Bis-ANS was used to investigate structural
changes in tubulin monomers and dimers during time- and temperature-dependent decay.39,40
Figure 11.2.12 Vinblastine, BODIPY® FL conjugate (BODIPY® FL vinblastine, V12390).
DCVJ (4-(dicyanovinyl)julolidine; D3923), which binds to a specific
site on the tubulin dimer,41 has been reported to be a useful probe for
following polymerization of tubulin in live cells.42 DCVJ staining in live
cells is mostly blocked by cytochalasin D.43 Additionally, DCVJ emits
strong green fluorescence upon binding to bovine brain calmodulin.44
The hydrophobic surfaces of tubulin have also been investigated with the
environment-sensitive probes nile red 45 (N1142) and prodan 46 (P248).
Probes for Other Cytoskeletal Proteins
GFP- and RFP-Labeled Talin
Talin is a cytoskeletal protein that is concentrated in focal adhesions, linking integrins to the actin cytoskeleton either directly
or indirectly by interacting with vinculin and α-actinin. CellLight®
Talin-GFP and CellLight® Talin-RFP expression vectors (C10611,
C10612; Table 11.1; Figure 11.2.13) generate autof luorescent proteins fused to the C-terminal actin-binding domain of human talin
and incorporate all the generic advantages of BacMam 2.0 delivery
technology (BacMam Gene Delivery and Expression Technology—
Note 11.1). These CellLight® reagents have potential applications
in image-based high-content screening (HCS) assays of integrinmediated cell adhesion, as well as for general-purpose labeling of
cytoskeletal actin in live cells.
Anti–Glial Fibrillary Acidic Protein (GFAP) Antibody
The 50,000-dalton type-III intermediate filament protein known as
glial fibrillary acidic protein (GFAP) is a major structural component
of astrocytes and some ependymal cells.47 GFAP associates with the
calcium-binding protein annexin II2-p11(2) and S-100.48,49 Association
with these proteins together with phosphorylation regulates GFAP polymerization. Astrocytes respond to brain injury by proliferation (astrogliosis); one of the first events to occur during astrocyte proliferation is increased GFAP expression. Our anti-GFAP antibody (A21282)
and its Alexa Fluor® 488 and Alexa Fluor® 594 conjugates (A21294,
A21295; Figure 11.2.14) can be used to aid in the identification of cells
of glial lineage. Interestingly, antibodies to GFAP have been detected in
Figure 11.2.13 HeLa cell labeled with CellLight® Talin-GFP (C10611) and CellLight® Actin-RFP
(C10583) reagents.
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492
IMPORTANT
NOTICE:
The products
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covered
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Limited
Use Label
License(s).
Please
refer to thePlease
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onto
IMPORTANT
NOTICE
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described
in thisaremanual
are
by one
or more
Limited
Use Label
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refer
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the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.2 Probes for Tubulin and Other Cytoskeletal Proteins
individuals with dementia.50 In the central nervous system, anti-GFAP
antibody stains both astrocytes and ependymal cells. In the peripheral nervous system, Schwann cells, satellite cells and enteric glial cells
are stained; tumors of glial origin contain high amounts of GFAP. No
positive staining is observed in skin, connective tissue, adipose tissue,
lymphatic tissue, muscle, kidney, ureter, bladder or gastrointestinal
tract, including liver and pancreas. Our anti-GFAP antibody does not
cross-react with vimentin, which is frequently co-expressed in glioma
cells and some astrocytes, nor does it cross-react with Bergmann glia
cells, gliomas or other glial cell–derived tumors.
Anti-Desmin Antibody
Desmin, encoded by a gene belonging to the intermediate filament
protein gene family,51–53 is the main intermediate filament in mature
skeletal, cardiac and smooth muscle cells. Both striated and smooth
muscle cells can be labeled by an anti-desmin antibody, although not all
muscle tissue contains desmin (e.g., aorta smooth muscle). Identification
of desmin is useful in distinguishing habdomyosarcomas and leiomyosarcomas from other vimentin-positive sarcomas. We offer a mouse
IgG1 monoclonal anti-desmin antibody (A21283), which can be used
with our fluorescent secondary antibodies (Section 7.2, Figure 11.2.15)
as a marker for typing soft tissue sarcomas. Anti-desmin immunohistochemical staining in cell-block preparations may also be helpful in
distinguishing mesothelial cells from carcinoma.54
Figure 11.2.14 Rat brain cryosections were labeled with the red-fluorescent Alexa Fluor® 594
conjugate of anti–glial fibrillary acidic protein antibody (A21295). Nuclei were counterstained
with TOTO®-3 iodide (T3604, pseudocolored blue).
Anti-Synapsin I Antibody
Synapsin I, an actin-binding protein, is localized exclusively to
synaptic vesicles and thus serves as an excellent marker for synapses in
brain and other neuronal tissues.55,56 Synapsin I inhibits neurotransmitter release, an effect that is abolished upon its phosphorylation by
Ca 2+/calmodulin–dependent protein kinase II. For assaying the localization and abundance of synapsin I by western blot analysis, immunohistochemistry (Figure 11.2.16), enzyme-linked immunosorbent assay
(ELISA) or immunoprecipitation, we offer a polyclonal rabbit anti–synapsin I antibody as an affinity-purified IgG fraction (A6442). Although
raised against bovine synapsin I, this antibody also recognizes human,
rat and mouse synapsin I; it has little or no activity against synapsin II.
Figure 11.2.15 The intermediate filaments in bovine pulmonary artery endothelial cells, localized using our anti-desmin antibody (A21283), which was visualized with the Alexa Fluor® 647
goat anti–mouse IgG antibody (A21235). Endogenous biotin in the mitochondria was labeled
with Alexa Fluor® 546 streptavidin (S11225) and DNA in the cell was stained with blue-fluorescent DAPI (D1306, D3571, D21490).
Figure 11.2.16 Peripheral neurons in mouse intestinal cryosections were labeled with rabbit
anti–synapsin I antibody (A6442) and detected using Alexa Fluor® 488 goat anti–rabbit IgG
antibody (A11008). This tissue was counterstained with DAPI (D1306, D3571, D21490).
™
The
Probes
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
TheMolecular
Molecular
Probes®
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
IMPORTANT
NOTICE:described
The products
described
thiscovered
manual are
by oneLimited
or moreUse
Limited
Use
Label License(s).
to the
Appendix
IMPORTANT NOTICE
: The products
in this
manualinare
by covered
one or more
Label
License(s).
PleasePlease
referrefer
to the
Appendix
on on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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493
Chapter 11 — Probes for Cytoskeletal Proteins
Section 11.2 Probes for Tubulin and Other Cytoskeletal Proteins
REFERENCES
1. BMC Cancer (2006) 6:86; 2. J Am Chem Soc (1971) 93:2325; 3. J Am Chem Soc
(1988) 110:5917; 4. Tetrahedron (1986) 42:4451; 5. J Biol Chem (1994) 269:23399;
6. J Cell Biol (1991) 112:1177; 7. Pharmacol Ther (1984) 25:83; 8. Cancer Treat Rep (1978)
62:1219; 9. PLoS Biol (2008) 6:e209; 10. J Neurosci (2008) 28:2601; 11. J Neurochem
(2007) 102:1009; 12. J Biol Chem (2003) 278:8407; 13. Biochemistry (2002) 41:12436;
14. Biochemistry (2001) 40:11975; 15. Cell Motil Cytoskeleton (2001) 49:1; 16. J Biol
Chem (2000) 275:26265; 17. J Cell Biol (2000) 148:883; 18. Chem Biol (2000) 7:275;
19. Biotechniques (1998) 25:188; 20. Mol Biotechnol (2002) 21:241; 21. Mol Pharmacol
(2002) 62:1238; 22. Cancer Res (2002) 62:6864; 23. Mol Biol Cell (1995) 6:1215; 24. Mol
Pharmacol (2002) 62:1; 25. FEBS Lett (1997) 416:251; 26. J Biol Chem (1996) 271:14707;
27. Eur J Biochem (1997) 244:664; 28. J Neurochem (1996) 67:1688; 29. Biochemistry
(1994) 33:12665; 30. Acta Histochem (1993) 94:54; 31. Arch Biochem Biophys
(1993) 303:159; 32. Eur J Biochem (1987) 165:613; 33. J Biol Chem (1985) 260:2819;
34. Anal Biochem (2003) 315:49; 35. J Biol Chem (1984) 259:14647; 36. Biochemistry
(1994) 33:11900; 37. Biochemistry (1994) 33:11891; 38. Biochemistry (1986) 25:3536;
39. Biochemistry (1998) 37:4687; 40. Biochemistry (1995) 34:13367; 41. Cell (1990) 62:579;
42. Biochemistry (1989) 28:6678; 43. Immunol Lett (1992) 33:285; 44. J Biochem (Tokyo)
(1991) 109:499; 45. J Biol Chem (1990) 265:14899; 46. Eur J Biochem (1992) 204:127;
47. Neurochem Res (2000) 25:1439; 48. Biochem Biophys Res Commun (1995) 208:910;
49. Biochim Biophys Acta (1996) 1313:268; 50. J Neurol Sci (1997) 151:41; 51. Proc Natl
Acad Sci U S A (1976) 73:4344; 52. J Cell Sci (1977) 23:243; 53. EMBO J (1982) 1:1649;
54. Acta Cytol (2000) 44:976; 55. Science (1984) 226:1209; 56. J Cell Biol (1983) 96:1337.
DATA TABLE 11.2 PROBES FOR TUBULIN AND OTHER CYTOSKELETAL PROTEINS
Cat. No.
MW
Storage
Soluble
Abs
EC
Em
Solvent
Notes
B153
672.85
L
pH >6
395
23,000
500
MeOH
1, 2
342
28,000
450
pH 7
3
D1306
350.25
L
H2O, DMF
342
28,000
450
pH 7
3
D3571
457.49
L
H2O, MeOH
D3923
249.31
L
DMF, DMSO
456
61,000
493
MeOH
4
342
28,000
450
pH 7
3, 5
D21490
350.25
L
H2O, DMF
N1142
318.37
L
DMF, DMSO
552
45,000
636
MeOH
6
P248
227.31
L
DMF, MeCN
363
19,000
497
MeOH
7
P3456
853.92
F,D
MeOH, DMSO
228
30,000
none
MeOH
P7500
1023.89
FF,D,L
DMSO
504
66,000
511
MeOH
P7501
1098.98
FF,D,L
DMSO
565
121,000
571
MeOH
P22310
1319.28
FF,D,L
DMSO, EtOH
494
80,000
522
pH 9
V12390
1043.02
F,D,L
DMSO, DMF
503
83,000
510
MeOH
For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.
Notes
1. B153 is soluble in water at 0.1–1.0 mM after heating.
2. Bis-ANS (B153) bound to tubulin has Abs = 392 nm, Em = 490 nm and a fluorescence quantum yield of 0.23. (Biochemistry (1994) 33:11900)
3. DAPI undergoes an approximately 9-fold fluorescence enhancement on binding to polymerized tubulin. Abs = 345 nm, Em = 446 nm. (J Biol Chem (1985) 260:2819)
4. The absorption and fluorescence emission maxima of DCVJ (D3923) bound to tubulin are essentially the same as in methanol. (Biochemistry (1989) 28:6678)
5. This product is specified to equal or exceed 98% analytical purity by HPLC.
6. The fluorescence emission maximum of nile red (N1142) bound to tubulin is 623 nm. (J Biol Chem (1990) 265:14899)
7. The fluorescence emission maximum of prodan (P248) bound to tubulin is ~450 nm. (Eur J Biochem (1992) 204:127)
PRODUCT LIST 11.2 PROBES FOR TUBULIN AND OTHER CYTOSKELETAL PROTEINS
Cat. No.
Product
A21283
A21282
A21294
A21295
A6442
A11126
A21371
B153
C10598
C10599
C10611
C10612
C10613
C10614
D3923
D1306
D21490
D3571
N1142
P3456
P7501
P7500
P22310
P248
T34075
V12390
anti-desmin, mouse IgG1, monoclonal 131-15014 *1 mg/mL*
anti-GFAP (anti–glial fibrillary acidic protein, mouse IgG1, monoclonal 131-17719) *1 mg/mL*
anti-GFAP, Alexa Fluor® 488 conjugate (anti–glial fibrillary acidic protein, mouse IgG1, monoclonal 131-17719, Alexa Fluor® 488 conjugate) *1 mg/mL*
anti-GFAP, Alexa Fluor® 594 conjugate (anti–glial fibrillary acidic protein, mouse IgG1, monoclonal 131-17719, Alexa Fluor® 594 conjugate) *1 mg/mL*
anti-synapsin I (bovine), rabbit IgG fraction *affinity purified*
anti-α-tubulin (bovine), mouse IgG1, monoclonal 236-10501
anti-α-tubulin (bovine), mouse IgG1, monoclonal 236-10501, biotin-XX conjugate
bis-ANS (4,4’-dianilino-1,1’-binaphthyl-5,5’-disulfonic acid, dipotassium salt)
CellLight® MAP4-GFP *BacMam 2.0*
CellLight® MAP4-RFP *BacMam 2.0*
CellLight® Talin-GFP *BacMam 2.0*
CellLight® Talin-RFP *BacMam 2.0*
CellLight® Tubulin-GFP *BacMam 2.0*
CellLight® Tubulin-RFP *BacMam 2.0*
DCVJ (4-(dicyanovinyl)julolidine)
4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI)
4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI) *FluoroPure™ grade*
4’,6-diamidino-2-phenylindole, dilactate (DAPI, dilactate)
nile red
paclitaxel (Taxol® equivalent) *for use in research only*
paclitaxel, BODIPY® 564/570 conjugate (BODIPY® 564/570 Taxol®)
paclitaxel, BODIPY® FL conjugate (BODIPY® FL Taxol®)
paclitaxel, Oregon Green® 488 conjugate (Oregon Green® 488 Taxol®; Flutax-2)
prodan (6-propionyl-2-dimethylaminonaphthalene)
TubulinTracker™ Green (Oregon Green® 488 Taxol®, bis-acetate) *for live-cell imaging*
vinblastine, BODIPY® FL conjugate (BODIPY® FL vinblastine)
Quantity
The
MolecularProbes®
Probes Handbook:
Handbook: AA Guide
Probesand
andLabeling
LabelingTechnologies
Technologies
The
Molecular
Guide to
to Fluorescent
Fluorescent Probes
™
494
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
www.invitrogen.com/probes
thermofisher.com/probes
100 µL
100 µL
50 µL
50 µL
10 µg
50 µg
50 µg
10 mg
1 mL
1 mL
1 mL
1 mL
1 mL
1 mL
25 mg
10 mg
10 mg
10 mg
25 mg
5 mg
10 µg
10 µg
100 µg
100 mg
1 set
100 µg