Product datasheet
Anti-Rad21 antibody ab42478
1 Abreviews 5 Images
Overview
Product name
Anti-Rad21 antibody
Description
Rabbit polyclonal to Rad21
Tested applications
WB, ICC/IF, IP
Species reactivity
Reacts with: Mouse, Human
Predicted to work with: Rat, Chicken, Cow
Immunogen
Synthetic peptide conjugated to KLH derived from within residues 200 - 300 of Human
Rad21.Read Abcam's proprietary immunogen policy(Peptide available as ab42520.)
Positive control
This antibody gave a positive signal in the following lysates: HeLa Whole Cell, HEK293 Whole
Cell, A431 Whole Cell, Mouse Brain Tissue, Mouse Testis Tissue
Properties
Form
Liquid
Storage instructions
Store at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated
freeze / thaw cycles.
Storage buffer
Preservative: 0.02% Sodium Azide
Constituents: 1% BSA, PBS, pH 7.4
Purity
Immunogen affinity purified
Clonality
Polyclonal
Isotype
IgG
Applications
Our Abpromise guarantee covers the use of ab42478 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application
Abreviews
Notes
1
Application
WB
Abreviews
Notes
Use a concentration of 1 µg/ml. Detects a band of approximately 100 kDa
(predicted molecular weight: 75 kDa). Although the predicted band size is 75kDa
based on Swiss-prot data, a band of 120kDa has been previously observed. Mol
Cell Biol. 2002 Dec;22(23):8267-77 PMID: 12417729. The band seen at 100kDa
is also consistent with the banding pattern observed for other commercially
available antibodies to Rad21.
ICC/IF
Use a concentration of 1 µg/ml.
IP
Use at an assay dependent concentration.
Target
Function
Cleavable component of the cohesin complex, involved in chromosome cohesion during cell
cycle, in DNA repair, and in apoptosis. The cohesin complex is required for the cohesion of
sister chromatids after DNA replication. The cohesin complex apparently forms a large
proteinaceous ring within which sister chromatids can be trapped. At metaphase-anaphase
transition, this protein is cleaved by separase/ESPL1 and dissociates from chromatin, allowing
sister chromatids to segregate. The cohesin complex may also play a role in spindle pole
assembly during mitosis. Also plays a role in apoptosis, via its cleavage by caspase-3/CASP3
or caspase-7/CASP7 during early steps of apoptosis: the C-terminal 64 kDa cleavage product
may act as a nuclear signal to initiate cytoplasmic events involved in the apoptotic pathway.
Sequence similarities
Belongs to the rad21 family.
Domain
The C-terminal part associates with the head of SMC1A, while the N-terminal part binds to the
head of SMC3.
Post-translational
modifications
Cleaved by separase/ESPL1 at the onset of anaphase. Cleaved by caspase-3 and caspase-7
at the beginning of apoptosis. The cleavage by ESPL1 and caspase-3 take place at different
sites.
Phosphorylated; becomes hyperphosphorylated in M phase of cell cycle. The large dissociation
of cohesin from chromosome arms during prophase may be partly due to its phosphorylation by
PLK.
Cellular localization
Nucleus. Chromosome. Chromosome > centromere. Associates with chromatin. Before
prophase it is scattered along chromosome arms. During prophase, most of cohesin complexes
dissociate from chromatin probably because of phosphorylation by PLK, except at centromeres,
where cohesin complexes remain. At anaphase, it is cleaved by separase/ESPL1, leading to
the dissociation of the complex from chromosomes, allowing chromosome separation. Once
cleaved by caspase-3, the C-terminal 64 kDa cleavage product translocates to the cytoplasm,
where it may trigger apoptosis.
Anti-Rad21 antibody images
2
ab42478 (1/200) staining Rad21 in
assynchronous HeLa cells (green). Cells were
fixed in paraformaldehyde, permeabilized
with 0.5% Triton X-100 and counterstained
with DAPI in order to highlight the nucleus
Immunocytochemistry/ Immunofluorescence -
(red). for further experimental details please
Rad21 antibody (ab42478)
see Abreview.
Image courtesy of an Abreview submitted by Dr. Kirk
McManus, Univ. of Manitoba/Cancer Care MICB,
Canada
All lanes : Anti-Rad21 antibody (ab42478) at
1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma
cell line) Whole Cell Lysate
Lane 2 : HEK293 Human embryonic kidney
cell line Whole Cell Lysate
Lane 3 : A431 (Human epithelial carcinoma
cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Western blot - Rad21 antibody (ab42478)
Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG
(H+L) at 1/10000 dilution
Performed under reducing conditions.
Predicted band size : 75 kDa
Observed band size : 100 kDa
Additional bands at : 55 kDa (possible
cleavage fragment).
3
All lanes : Anti-Rad21 antibody (ab42478) at
1 µg/ml
Lane 1 : Brain (Mouse) Tissue Lysate
Lane 2 : Testis (Mouse) Tissue Lysate
Lysates/proteins at 10 µg per lane.
Secondary
Goat polyclonal to Rabbit IgG - H&L - PreAdsorbed (HRP) at 1/3000 dilution
Western blot - Rad21 antibody (ab42478)
developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 75 kDa
Observed band size : 120 kDa
Additional bands at : 55 kDa,85 kDa. We
are unsure as to the identity of these extra
bands.
Exposure time : 4 minutes
4
Rad21 was immunoprecipitated using 0.5mg
Hela whole cell extract, 5µg of Rabbit
polyclonal to Rad21 and 50µl of protein G
magnetic beads (+). No antibody was added
to the control (-).
The antibody was incubated under agitation
with Protein G beads for 10min, Hela whole
cell extract lysate diluted in RIPA buffer was
added to each sample and incubated for a
further 10min under agitation.
Proteins were eluted by addition of 40µl SDS
Immunoprecipitation - Anti-Rad21 antibody
loading buffer and incubated for 10min at
(ab42478)
70oC; 10µl of each sample was separated on
a SDS PAGE gel, transferred to a
nitrocellulose membrane, blocked with 5%
BSA and probed with ab42478.
Secondary: Goat polyclonal to mouse IgG
light chain specific (HRP) at 1/5000 dilution.
Band: 100kDa: Rad21; Non specific - 50kDa:
We are unsure as to the identity of this extra
band. Although the predicted band size is
75kDa based on Swiss-prot data, a band of
120kDa has been previously observed. Mol
Cell Biol. 2002 Dec;22(23):8267-77 PMID:
12417729. The band seen at 100kDa is also
consistent with the banding pattern observed
for other commercially available antibodies to
Rad21.
ICC/IF image of ab42478 stained human
HeLa cells. The cells were PFA fixed (10
min), permabilised in TBS-T (20 min) and
incubated with the antibody (ab42478,
1µg/ml) for 1h at room temperature. 1%BSA /
10% normal goat serum / 0.3M glycine was
used to quench autofluorescence and block
non-specific protein-protein interactions. The
Immunocytochemistry/ Immunofluorescence -
secondary antibody (green) was Alexa Fluor®
Rad21 antibody (ab42478)
488 goat anti-rabbit IgG (H+L) used at a
1/1000 dilution for 1h. Alexa Fluor® 594 WGA
was used to label plasma membranes (red).
DAPI was used to stain the cell nuclei (blue).
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