ab170196 –
RSK1 p90 (Total + pSer380)
In-Cell ELISA Kit
(Fluorescent)
Instructions for Use
For measuring in high throughput levels of RSK1 p90 total protein and
phosphorylated at Ser380.
This product is for research use only and is not intended for diagnostic
use.
Version1 Last Updated26 April 2013
Table of Contents
INTRODUCTION
1.
BACKGROUND
2.
ASSAY SUMMARY
2
3
GENERAL INFORMATION
3.
PRECAUTIONS
4.
STORAGE AND STABILITY
5.
MATERIALS SUPPLIED
6.
MATERIALS REQUIRED, NOT SUPPLIED
7.
LIMITATIONS
8.
TECHNICAL HINTS
4
4
5
6
7
7
ASSAY PREPARATION
9.
REAGENT PREPARATION
10. SAMPLE PREPARATION
8
10
ASSAY PROCEDURE
11.
12.
ASSAY PROCEDURE
CALCULATIONS
13
16
DATA ANALYSIS
13. TYPICAL DATA
14. TYPICAL SAMPLE VALUES
15. SPECIES REACTIVITY
16. ASSAY SPECIFICITY
17
18
18
19
RESOURCES
17. TROUBLESHOOTING
18. NOTES
24
25
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1
INTRODUCTION
1. BACKGROUND
Abcam’s RSK1 p90 Total and pSer380 In-Cell ELISA (ICE) kit is
designed to measure levels of RSK1 p90 total protein and
phosphorylated at serine 380 in cultured cells.
RSK1 p90 is a 90kDa ribosomal S6 kinase that belongs to a family of
serine and threonine kinases. These kinases include a binding site for
extracellular signal-related kinases (ERKs) at its carboxy-terminus.
Several residues including Thr359, Ser363, Ser380, and Thr573 are
involved in downstream kinase activation pathways. In response to
many growth factors, neurotransmitters, and hormones, RSK1 p90 is
activated through phosphorylation by MAPK, PI3K and by autophosphorylation.
Upon
phosphorylation
at
Ser380,
PDK1
phosphorylates Ser221, resulting in full activation of RSK1 p90. This
pathway is known for promoting cell growth, regulating apoptosis,
chemotherapeutic drug resistance, and cellular senescence.
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2
INTRODUCTION
2. ASSAY SUMMARY
Seed cells in a microwell culture plate. Fix with 4% paraformaldehyde
for 10 minutes and wash. Store overnight in PBS with azide
Treat cells with 1X Quenching Buffer at RT for 15 minutes and wash
Incubate with Antigen Retrieval Buffer at 80°C for 15 minutes and
wash
Permeabilize/block cells for 2 hours at RT.
Incubate cells with primary antibodies overnight at 4°C or for 2 hours at
RT and wash.
Incubate cells with secondary antibodies for 2 hours at RT and wash
Add fluorogenic substrates
Read on spectrophotometer
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3
GENERAL INFORMATION
3. PRECAUTIONS
Please read these instructions carefully prior to beginning the
assay.
All kit components have been formulated and quality control tested to
function successfully as a kit. Modifications to the kit components or
procedures may result in loss of performance.
4. STORAGE AND STABILITY
Store kit at 4ºC immediately upon receipt.
Refer to list of materials supplied for storage conditions of individual
components. Observe the storage conditions for individual prepared
components in sections 9 & 10.
Upon receipt spin down the contents of the primary and secondary
antibodies and store upright. Store fluorescent substrate cocktail
protected from the light.
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4
GENERAL INFORMATION
5. MATERIALS SUPPLIED
Amount
Storage
Condition
(Before
Preparation)
10X Phosphate Buffered Saline (PBS)
400X Tween - 20 (20% solution)
100 mL
4 mL
4ºC
4ºC
100X Triton X-100 (10% solution)
10X Blocking Buffer
Antigen Retrieval Buffer
100X RSK1 p90 (total and pSer380) Primary
Antibody Cocktail
1000X AP-Labeled Secondary antibody
(anti-Rabbit IgG)
1000X HRP-Labeled Secondary antibody
(anti-Mouse IgG)
400X Fluorescent Substrate Cocktail
Fluorescent Substrate Buffer
8000X Hydrogen Peroxide
10X Quenching Solution
Janus Green Stain
1.25 mL
10 mL
25 mL
4ºC
4ºC
4ºC
120 µL
4ºC
20 µL
4ºC
20 µL
4ºC
50 µL
12 mL
50 µL
4ºC
4ºC
4ºC
4ºC
4ºC
Item
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1.5 mL
11 mL
5
GENERAL INFORMATION
6. MATERIALS REQUIRED, NOT SUPPLIED
These materials are not included in the kit, but will be required to
successfully utilize this assay:

96 or 384-well amine coated plate(s).

20% paraformaldehyde.

Nanopure water or equivalent.

Water bath.

Microplate-adaptable heating block.

Multi and single channel pipettes.

0.5 M HCl (optional for Janus Green cell staining procedure).

Optional humid box for overnight incubation step.

Optional plate shaker for all incubation steps.

Fluorescent spectrophotometer to view AP and HRP signals:
RSK1 p90
Total
RSK1 p90
pSer380
Absorption
Max (nm)
Emission
Max (nm)
Secondary
Label
555 ± 15
595 ± 5
HRP
360 ± 5
449 ± 10
AP
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6
GENERAL INFORMATION
7. LIMITATIONS

Assay kit intended for research use only. Not for use in diagnostic
procedures.

Do not use kit or components if it has exceeded the expiration date
on the kit labels.

Do not mix or substitute reagents or materials from other kit lots or
vendors. Kits are QC tested as a set of components and
performance cannot be guaranteed if utilized separately or
substituted.
8. TECHNICAL HINTS

Avoid foaming
components.

Avoid cross contamination of samples or reagents by changing tips
between sample and reagent additions.

Ensure plates are properly sealed or covered during incubation
steps.

Complete removal of all solutions and buffers during wash steps.

During development of this assay we have observed edge effects
after treatment with Antigen Retrieval Buffer. Use perimeter wells
of the plate as control wells (primary antibody omitted).
Regardless, it is required to leave at minimum one well from which
the primary antibodies are excluded to determine background
signals of the assay.
or
bubbles
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when
mixing
or
reconstituting
7
ASSAY PREPARATION
9. REAGENT PREPARATION
Equilibrate all reagents to room temperature (18-25°C) prior to use.
9.1
1X PBS
Prepare 1X PBS by diluting 100 mL of 10X PBS in 900 mL
of nanopure water or equivalent. Mix well. Store at room
temperature.
9.2
1X Wash Buffer
Prepare 1X Wash Buffer by diluting 1.25 mL of
400X Tween-20 in 500 mL of 1X PBS. Mix well. Store at
room temperature.
9.3
8% Paraformaldehyde Solution
Immediately prior to use prepare 8% Paraformaldehyde
Solution in 1X PBS. To make 8% Paraformaldehyde,
combine 6 mL of 1X PBS and 4 mL of
20% Paraformaldehyde. Note – Paraformaldehyde is toxic
and should be prepared and used in a fume hood. Dispose
of paraformaldehyde according to local regulations.
9.4
1X Quenching Solution
Prepare 1X Quenching solution by diluting 1.2 mL of
10X Quenching Solution in 10.8 mL of nanopure water. Mix
well and store at room temperature.
9.5
Antigen Retrieval Buffer
Pre-heat Antigen Retrieval Buffer to 80⁰C in a water bath
before starting assay. A microplate heating block should also
be pre-heated to 80⁰C.
9.6
1X Permeabilization/Blocking Solution
Immediately
prior
to
use
prepare
1X Permeabilization/Blocking Solution by diluting 500 µL of
100X Triton X-100, 5 mL of 10X Blocking Buffer in 44.5 mL
of 1X PBS. Mix well.
9.7
1X Primary Antibody Cocktail Solution
Prepare a Primary Antibody Cocktail Solution by diluting
100X of RSK1 p90 (total and pSer380) Primary Antibody
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8
ASSAY PREPARATION
Cocktail
into
an
appropriate
volume
of
1X Permeabilization/Blocking Solution (i.e. 12 mL of
1X Permeabilization/Blocking solution + 120 µL of the
100X Primary antibody cocktail). Note – This 1X cocktail
should only be made immediately prior to adding to
microplate
9.8
1X Secondary Antibody Cocktail Solution
Prepare 1X Secondary Antibody Cocktail Solution by diluting
12 µL of 1000X HRP-Labeled anti-mouse antibody and
12 µL of 1000X AP-Labeled anti-rabbit antibody in 12 mL
1X Permeabilization/Blocking solution. Note – This
1X cocktail should only be made immediately prior to adding
to microplate.
9.9
1X Development Solution
Prepare 1X Development Solution by diluting 30 µL of
400X Fluorescent substrate cocktail and 1.5 µL of
8000X Hydrogen Peroxide in 12 mL of Fluorescent substrate
buffer. Note – Development solution should be made
immediately prior to adding to microplate.
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9
ASSAY PREPARATION
10. SAMPLE PREPARATION
General Sample information:
●
The protocol below is described for a 96-well plate. If performing
assay on a 384-well plate, adjust volumes accordingly.
10.1 Preparation of adherent cells
10.1.1 Seed adherent cells directly into an amine coated
plate and allow them to attach for >6 hours or
overnight. It is advised to seed in a 100 µL volume
of the same media used to maintain the cells in bulk
culture. The optimal cell seeding density is cell type
dependent. The goal is to seed cells such that they
are just reaching confluency (but not over-confluent)
at the time of fixation. As an example, HeLa cells
may be seeded between 50,000 and 100,000 cells
per well and cultured overnight for fixation the
following day. Note = It is advisable to add three
blank wells (with no cells) when using media other
than MEM or DMEM.
10.1.2 The attached cells can be treated if desired with a
drug of interest.
10.1.3 Fix cells by adding a final concentration of 4%
Paraformaldehyde Solution. This can be achieved
by one of two means: (1) Add an equal volume of
8% Paraformaldehyde Solution to the culture volume
(e.g. add 100 µL 8% Paraformaldehyde to a well
with 100 µL media) or (2) gently remove culture
media from the wells and replace with 100 µL 4%
Paraformaldehyde Solution.
10.1.4 Incubate for 10-20 minutes at room temperature.
10.1.5 Gently remove the Paraformaldehyde Solution from
the plate and wash the plate three times briefly with
1X PBS. For each wash, rinse each well of the plate
with 200 µL of 1X PBS.
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10
ASSAY PREPARATION
10.1.6 Add 100 µL of 1X PBS with 0.02% sodium azide and
store the plate overnight.
Sodium azide will
preserve the plate for long storage and it will
decrease the peroxidase background normally found
on fixed cells.
10.1.7 Remove 1X PBS with 0.02% sodium azide and add
100 µL of 1X Quenching solution. Incubate for 10
minutes at room temperature.
The quenching
solution will decrease the phosphatase background
that may remain in fixed cells.
10.1.8 Wash the plate 3 times with 1X PBS and proceed
immediately to section 11.
10.2
Preparation of suspension cells
10.2.1 To ensure efficient cross-linking of the suspension
cells to the amine plate, cells must be grown and
treated in a different plate or dish of choice with no
more than 10% FBS. The cell seeding density of the
amine plate is cell type-dependent. As an example,
HL-60 and Jurkat cells should be seeded,
respectively, at 300,000 and 200,000 cells per well
in 100 µLof media.
10.2.2 When treatment is completed, transferred treated
suspension cells to an amine plate.
10.2.3 Centrifuge the microtiter plate at 500 x g for 5-10
minutes.
10.2.4 Fix cells by adding a final concentration of
4% Paraformaldehyde Solution.
This can be
achieved by overlaying an equal volume of
8% Paraformaldehyde Solution to the culture volume
(e.g. add 100 µL 8% Paraformaldehyde to a well
with 100 µL media).
10.2.5 Centrifuge the microtiter plate again at 500 x g for
5-10 minutes.
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11
ASSAY PREPARATION
10.2.6 Continue in the fixation for a total of 15 - 20 minutes.
10.2.7 Gently remove the Paraformaldehyde Solution from
the plate and wash the plate three times briefly with
1X PBS. For each wash, rinse each well of the plate
with 200 µL of 1X PBS.
10.2.8 Add 100 µL of 1X PBS with 0.02% sodium azide and
store the plate overnight.
Sodium azide will
preserve the plate for long storage and it will
decrease the peroxidase background normally found
on fixed cells.
10.2.9 Remove 1X PBS with 0.02% sodium azide and add
100 µL of 1X Quenching solution. Incubate for 10
minutes at room temperature.
The quenching
solution will decrease the phosphatase background
that may remain in fixed cells
10.2.10 Wash the plate 3 times with 1X PBS and proceed
immediately to section 11.
Note – The plate should not be allowed to dry at any point during or
before the assay. Both paraformaldehyde and sodium azide are toxic,
handle with care and dispose of according to local regulations.
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12
ASSAY PROCEDURE
11. ASSAY PROCEDURE
●
It is recommended to use a plate shaker (~200 rpm) during all
incubation steps. Any step involving removal of buffer or
solution should be followed by blotting the plate gently
upside down on a paper towel before refilling wells. Unless
otherwise noted, incubate at room temperature.
●
It is recommended to assay all samples in duplicate.
11.1. Ensure that cellular peroxidase and phosphatase
background (see section 10) have been quenched before
continuing with the assay procedure.
11.2. Remove 1X PBS and add 200 µL of pre-heated Antigen
Retrieval Buffer to each well of the plate. Incubate for 15
minutes at 80⁰C.
11.3. Remove the Antigen Retrieval Buffer and wash the plate
3 times briefly with 1X PBS. For each wash, rinse each
well of the plate with 200 µL.
11.4. Remove
1X
PBS
and
add
200
µL
of
1X Permeabilization/Blocking Solution to each well of the
plate. Incubate for 2 hours at room temperature.
11.5. Remove Permeabilization/Blocking Solution and add
100 µL 1X Primary Antibody Cocktail Solution to each well
of the plate. Incubate for 2 hours at room temperature or
overnight at 4°C. Note – To determine the background
signal it is essential to omit primary antibody from at least
one well containing cells for each experimental condition.
11.6. Remove Primary Antibody Cocktail Solution and wash the
plate 3 times briefly with 1X Wash Buffer. For each wash,
rinse each well of the plate with 200 µL of 1X Wash Buffer.
Do not remove the last wash until the secondary antibody
cocktail solution has been prepared.
11.7. Remove 1X Wash Buffer and add 100 µL 1X Secondary
Antibody Solution to each well of the plate. Incubate
2 hours at room temperature.
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13
ASSAY PROCEDURE
11.8. Remove 1X Secondary Antibody Cocktail Solution and
wash 3 times briefly with 1X Wash Buffer. For each wash,
rinse each well of the plate with 200 µL of 1X Wash Buffer.
11.9. Remove 1X Wash Buffer and wash 3 times briefly with
1X PBS. For each wash, rinse each well of the plate with
200 µL of 1X PBS. Do not remove the last wash until
development solution has been prepared.
11.10. Remove 1X PBS and add 100 µL per well of
1X Development Solution and immediately begin recording
if performing a kinetic assay or incubate for 30 – 60 minutes
if performing an end point assay. Spectrophotometer
settings should be as follows:
End Point
Mode:
Kinetic
Excitation
spectra
Emission
spectra:
AP substrate = 360 ± 5 nm
HRP substrate = 555 ± 15 nm
AP substrate = 449 ± 10 nm
HRP substrate = 595 ± 5nm
AP signal = 45 – 60 min
up to 60 min
HRP signal = 30 – 60 min
n/a
1 - 5 min
Time:
Interval:
Shaking:
Shake between
readings
n/a
11.11. Remove the Development solution and wash the plate 3
times with 1X PBS
11.12. Remove PBS and add 100 µL of Janus Green Stain to each
well of the plate. Incubate plate for 10 minutes at room
temperature. Note – The RFU signal should be normalized
to the Janus Green staining intensity to account for
differences in cell density.
11.13. Remove the dye and wash the plate 5 times in deionized
water or until excess dye is removed.
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14
ASSAY PROCEDURE
11.14. Remove last water wash, blot to dry, add 100 µL of 0.5 M
HCl to each well of the plate and incubate for 10 minutes in
a plate shaker.
11.15. Measure OD 595 nm using a standard microplate
spectrophotometer.
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15
TYPICAL DATA
12. CALCULATIONS
12.1 Background Subtraction
Determine the raw RFU signal values for each substrate.
Subtract the mean background values from all other RFU
experimental values respectively.
12.2 Janus Green normalization
Divide the background subtracted RFU intensities (from
12.1) by the Janus Green value of the corresponding well.
The result is the “normalized intensity”.
12.3 Normalization of Phospho signal to total protein.
Divide the RSK1 p90 pSer380 normalized intensity by the
total RSK1 p90 normalized intensity.
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16
TYPICAL DATA
13. TYPICAL DATA
TYPICAL STANDARD CURVE – Data provided for demonstration
purposes only.
Assay performance was tested using HeLa cells treated with 200nM of
phorbol 12-myristate 13-acetate (PMA) also known as 12-OTetradecanoylphorbol 13-acetate (TPA) for 1 hour in 0F-HGDMEM
media after overnight serum starvation. PMA is a potent activator of
protein kinase C (PKC), which phosphorylates RSK1 p90 at Ser380.
Figure 1 shows dynamic range of the assay on amine coated plates.
Linearity of signal is observed between 25,000 -100,000 cells per well.
Cells/Well
0
1,563
3,125
6,250
12,500
25,000
50,000
100,000
RSK1 p90 Total
RFU (Ex 555/ Em 595)
0
1,366
2,571
5,146
8,150
13,591
19,018
24,536
RSK1 p90 pSer380
RFU (Ex 360/ Em 450)
0
1,534
2,627
5,599
9,984
14,576
19,119
24,815
Figure 1. Dynamic range of RSK1 p90 total and pSer380. HeLa cells were
seeded on amine coated plates within the working range of the assay the day
before fixation. Levels of total RSK1 p90 and phosphorylated protein at Ser380
were measured in PMA treated and untreated cells.
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17
TYPICAL DATA
14. TYPICAL SAMPLE VALUES
PRECISION
The coefficient of the intra-assay variation for this assay kit on HeLa
cells treated with PMA is typically 4.0% for RSK1 p90 and 5.3% for
RSK1 p90 pSer380. The assay was also found to be highly robust
with a mean Z factor from multiple cell densities of 0.780 for RSK1 p90
(12,000 – 100,000 cells/well) and 0.778 for RSK1 p90 pSer380
(12,000 – 100,000 cells/well).
15. SPECIES REACTIVITY
This kit detects RSK1 p90 total protein and pSer380 in human and
mouse samples. Cross reactivity to mouse samples was determined
with the mouse cell line NIH3T3 treated with 50 ng/mL of PDGF A/B
recombinant protein for 30 minutes after overnight serum starvation.
Figure 2 shows performance of this kit on NIH3T3 PDGF treated
against BSA control
Figure 2. Mouse Reactivity of RSK1 p90 total and pSer380 ICE assay.
Levels of total RSK1 p90 and phosphorylated protein at Ser380 were
measured in serum-starved NIH3T3 cells that were treated with PDGF
recombinant protein at 50 ng/mL in 1% BSA. The relative levels of Total RSK1
p90 (left) and RSK1 p90 pSer380 (middle) are shown after background
subtraction. Specific phosphorylation was calculated by determining the ratio
of pSer380 to Total RSK1 p90 levels after normalization (right).
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18
TYPICAL DATA
16. ASSAY SPECIFICITY
Confidence in antibody specificity is critical to ICE data interpretation;
therefore the primary antibodies in this kit were validated by ICE and
Immunocytochemistry (ICC) with the use of lambda phosphatase and
by Western blot with the presence of signal at the correct band size.
RSK1 p90 pSer380 was phosphorylated with PMA treatment, followed
by an artificial dephosphorylation with the use of lambda phosphatase
(LPP) after fixation and permeabilization on the ICE and ICC platform
and on western blot after cell lysis with a non-ionic detergent but
before the addition of SDS-PAGE buffer.
Figure 3. Specificity of Signal by In-Cell Elisa. Levels of total RSK1 p90 and
phosphorylated protein at Ser380 were measured in DMSO and PMA treated
cells. HeLa cells were seeded on amine coated plates within the working range
of the assay and serum starved overnight prior to 1 hour treatment with 200nM
PMA or DMSO. Specificity of phosphorylation was determined by treating
PMA-treated HeLa cells with LPP at 4,000 units/mL for 45 minutes at 40⁰C
immediately after permeabilization. The relative levels of Total RSK1 p90
(Left) and RSK1 p90 pSer380 (Middle) are shown after background subtraction
and normalization with Janus green. Specific Phosphorylation was calculated
by determining the ratio of normalized pSer380 to normalized Total RSK1 p90
(Right).
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19
TYPICAL DATA
Figure 4. Specificity of Signal by Immunocytochemistry. HeLa cells were
seeded on glass coverslips and allowed to adhere for a few hours. Cells were
then serum starved overnight and treated the next day with DMSO (A) or
200nM PMA (B and C). Levels of RSK1 p90 total and phosphorylated protein
at Ser380 were measured following this protocol. The specificity of signal was
determined by treating PMA-treated cells with 4,000 units/mL LPP prior to the
assay (C). The total RSK1 p90 antibody was labeled with GAM-488 (green)
whereas the RSK1 p90 pSer380 antibody was labeled with GAR-594 (red).
The nucleus was counterstained with DAPI. The panels show up-regulation of
phosphorylation levels due to PMA treatment and overlap of the total and
phospho signal shown by the yellow color (B) that is not seen in the DMSO
treated cells (A). The PMA induced phosphorylation was removed after
treatment with LPP (C).
Figure 5. Verification of mouse anti-RSK1 p90 total
protein signal by Western Blot. Western blot was run
on an 8% acrylamide gel. Samples were loaded from left
to right: (1) 100 ng of recombinant RSK1 p90 (rRSK1
p90) ab60880, (2) 40 µg of HeLa lysate. Blocking and
secondary antibody incubation steps were carried out in
5% milk, 20 mM Tris-HCl, 0.1% TWEEN-20. Primary
incubation steps were carried out in 1% milk, 20 mM TrisHCL, 0.1% TWEEN-20.
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20
TYPICAL DATA
Figure 6. Verification of rabbit anti-RSK1 p90
pSer380 signal by Western Blot. Western blot was
run on a 4-20% gradient acrylamide gel. Gel was
loaded from left to right: (1) 100ng of recombinant
RSK1 p90 (rRSK1 p90) ab60880, (2) 4 0µg of
Hek293T cells treated with DMSO only or (3)
Hek293T cells treated with 200 nM PMA (right).
Blocking and secondary antibody incubation steps
were carried out in 5% milk, 20 mM Tris-HCl, 0.1%
TWEEN-20. Primary incubation steps were carried
out in 1% milk, 20 mM Tris-HCL, 0.1% TWEEN-20.
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21
RESOURCES
FREQUENTLY ASKED QUESTIONS
How many cells do I seed per well?
The cell seeding density varies by cell type and depends both on the
cell size and the abundance of the target protein. The cell seeding will
likely need to be determined experimentally by microscopic cell density
observation of serially diluted cells. For adherent cells, prepare a serial
dilution of the cells in a plate and allow them to attach prior to
observation. The goal is to have cells that are just confluent at the
time of fixation. Overly confluent cells may have compromised viability
and tend to not adhere as well to the plate. Under-seeded cells may
yield too low a signal, depending on the analyte. Keep in mind that
drug treatments or culture conditions may affect cell density/growth.
Do I have to use an amine-coated microplate?
We have tested black wall amine and cell culture treated microplates
and found that amine coated plates improve reproducibility and
specificity in comparison to standard plates. In addition, multiple cell
types appear to have the most favorable growth and even seeding on
amine plates. The assay performance is only guaranteed with amine
plates.
A treatment causes cell detachment. Is there a way to prevent the loss
of detaching cells?
Loss of floating cells can be easily prevented by inserting two
centrifugation steps into the protocol: (1) Immediately prior the addition
of Paraformaldehyde Solution centrifuge the microtiter plate at 500 x g
for 5-10 minutes, (2) Immediately after the addition of
Paraformaldehyde Solution centrifuge the microtiter plate again at 500
x g for 5-10 minutes. Continue in the fixation for a total of 15 - 20
minutes.
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22
RESOURCES
I grow my cells in 15% FBS, will this interfere with the cell fixation?
Culture media containing up to 15% fetal serum does not interfere with
the cell fixation and cross-linking to the plate on adherent cells.
How do I measure the assay background?
It is essential to omit primary antibody in at least one well (3 wells
recommended) to provide a background signal for the experiment
which can be subtracted from all measured data. This should be done
for each experimental condition. It is also recommended to include at
least one well without cells to provide primary antibody background
particularly when cells are grown on RPMI media.
Is Janus Green normalization necessary?
Janus Green is a whole-cell stain that is useful to determine if a
decrease in IR intensity in a well is due to a relevant down-regulation
or degradation of the target analyte or if it is a function of decreased
cell number (e.g. due to cytotoxic effect of a treatment). As such it is
not a required readout, but it is useful in the analysis to determine a
normalized intensity value.
I don’t want to use azide to quench the endogenous peroxidase signal
on fixed cells. Is there another choice?
In the absence of azide, the endogenous peroxidase signal may be
quenched with a 20 minute treatment of 0.6% Hydrogen Peroxide in
PBS. Following this treatment, cells must be washed thoroughly with
PBS before proceeding with the rest of the experiment.
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23
RESOURCES
17. TROUBLESHOOTING
Problem
Cause
Solution
Endogenous, cellular
alkaline
phosphatase
Use quenching solution
before the assay procedure.
High AP
background
Growth media
High HRP
background
Endogenous cellular
Peroxidase activity
RPMI induces high AP
background in the absence
of cells. Perform assay with
an alternative media or
wash cells in the presence
of phosphatase inhibitors
prior to fixation.
Incubate the cells overnight
with PBS+0.02% azide or
Incubate the cells for 20
minutes with 0.6%
Hydrogen Peroxide in
PBS prior the assay
procedure.
Too brief incubation
times
Ensure sufficient incubation
times
Inadequate reagent
volumes or improper
dilution
Check pipettes and ensure
correct preparation
Insufficient cells
Increase seeding density of
cells; goal is newly
confluent cells at time of
fixation.
Cell detachment
Refer to frequently asked
questions
Low Signal
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24
RESOURCES
18. NOTES
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25
RESOURCES
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26
UK, EU and ROW
Email: technical@abcam.com | Tel: +44-(0)1223-696000
Austria
Email: wissenschaftlicherdienst@abcam.com | Tel: 019-288-259
France
Email: supportscientifique@abcam.com | Tel: 01-46-94-62-96
Germany
Email: wissenschaftlicherdienst@abcam.com | Tel: 030-896-779-154
Spain
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