ab156029
AKT1 Total and
pSer473 Human In-Cell
ELISA Kit (Fluorescent)
Instructions for Use
For measuring in high throughput the levels of AKT1 total and
phosphorylated protein at Ser473 in human cell lines.
This product is for research use only and is not intended for
diagnostic use.
Table of Contents
1.
Introduction………………………………………………………….2
2.
Assay Summary…………………………………………………….4
3.
Kit Contents………………………………………………………….5
4.
Storage and Handling………………………………………………6
5.
Additional Materials Required……………………………………..6
6.
Preparation of Reagents…………………………………………...6
7.
Sample Preparation………………………………………………...8
8.
Assay Procedure…………………………………………………..10
9.
Data Analysis………………………………………………………13
10. Assay Performance and Specificity……………………………..13
11. Frequently Asked Questions……………………………………..17
12. Troubleshooting……………………………………………………21
1
1. Introduction
Principle: Abcam’s AKT1 total and pSer473 In-Cell ELISA (ICE)
assay uses quantitative immunocytochemistry to measure the levels
of AKT1 total and phosphorylated protein at Ser473 in cultured cells.
Cells are fixed in a microplate and targets of interest are detected
with highly specific, well-characterized monoclonal antibodies.
Relative target levels are quantified using secondary antibodies
conjugated to either horseradish peroxidase (HRP) or alkaline
phosphatase (AP) which generate signal through the use of two
spectrally distinct fluorogenic substrates. Fluorescence is measured
using a standard fluorescent spectrophotometer.
Optionally,
antibody signal intensity can be normalized to the total cell stain
Janus Green.
Background: Akt is a serine, threonine protein kinase critical in
cellular
metabolism,
glucose
uptake,
protein
synthesis,
cell
proliferation, growth, apoptosis, survival, angiogenesis, migration
and invasion.
It acts downstream of the phosphatidylinositol 3
kinase (PI3K) and it mediates the effects of several growth factors
such as platelet-derived growth factor, epidermal growth factor and
insulin growth factor. It is activated by phosphorylation on Ser473,
Thr308 and Tyr474 and when active it phosphorylates transcription
factors (FOXO1), kinases (GSK-3, Raf-1, ASK, Chk1) and other
signaling proteins (Bad, MDM2). There are three Akt isoforms (Akt1,
Akt2 and Akt3) which share 80% sequence identity also known as
PKBα, PKBβ and PKBγ. Akt has been shown to have a role in
2
metabolism, apoptosis and proliferation and therefore it has been
proposed to be the candidate “Warburg Kinase”.
Akt is the most frequently activated oncoprotein in human cancers.
As a target for cancer therapy it has gain interest in highly
chemoresistant tumors.
Defects in AKT1 are a cause of
susceptibility to breast cancer (BC) [MIM:114480], colorectal cancer
(CRC) [MIM:114500] and ovarian cancer [MIM:604370]. Akt is also
intensively studied in in-vitro and in-vivo models of type II diabetes.
Akt also has been proposed to play a role in diseases mediated by
macrophage
innate
immunity
such
as
rheumatoid
arthritis,
atherosclerosis, diabetes, obesity, and osteoporosis and hence
could become a future therapeutic target in these areas.
In-Cell ELISA (ICE) technology is used to perform quantitative
immunocytochemistry of cultured cells.
The technique generates
quantitative data with specificity similar to Western blotting, but with
much greater quantitative precision and higher throughput due to the
greater dynamic range and linearity of fluorescence detection and
the ability to run up to 96 samples in parallel. This method rapidly
fixes the cells in situ, stabilizing the in vivo levels of proteins and
their post-translational modifications, and thus essentially eliminates
changes during sample handling, such as preparation of protein
extracts. Finally, the signal can be normalized to cell amount,
measured by the provided Janus Green whole cell stain, to further
increase the assay precision.
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2. Assay Summary
Seed cells into a microplate.
Fix cells with 4% paraformaldehyde for 10 minutes and wash with
PBS. Store overnight in PBS with azide
Treat cells with 1X Quenching buffer for 10 minutes and wash with
PBS
Permeabilize the cells with 100 µL/well of permeabilization buffer.
Incubate for 30 minutes at RT and discard.
Add 200 µL/well of Blocking buffer, incubate for 2 hours at RT and
discard.
Add primary antibodies diluted in blocking buffer for 2 hours at RT or
overnight at 4°C and wash with PBST.
Incubate cells for 2 hours at RT with secondary antibodies diluted in
Blocking Buffer and wash with PBST
Add fluorogenic substrates and read on spectrophotometer.
3. Kit Contents
4
Items
Quantity
10X Phosphate Buffered Saline (PBS)
100 mL
100X Triton X-100
1.25 mL
400X Tween – 20
2 mL
10X Blocking Buffer
15 mL
100X Mouse Anti Akt-1 Primary Antibody
120 µL
100X Rabbit Anti Akt-1 pS473 Primary
Antibody
120 µL
1000X AP-Labeled Secondary antibody
(anti-Mouse IgG)
20 µL
1000X HRP-Labeled Secondary antibody
(anti-Rabbit IgG)
20 µL
200X Fluorescent Substrate Cocktail
70 µL
Fluorescent Substrate Buffer
12 mL
8000X Hydrogen Peroxide
50 µL
10X Quenching Solution
1.5 mL
Janus Green Stain
17 mL
4. Storage and Handling
Upon receipt spin down the contents of all vials with less than 1mL of
volume. Store all components upright at 4°C.
This kit is stable for
at least 6 months from receipt.
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5. Additional Materials Required

Fluorescent spectrophotometer.

96 or 384-well amine coated plate(s).

20% paraformaldehyde.

Nanopure water or equivalent.

Multi and single channel pipettes.

0.5 M HCl (optional for Janus Green cell staining procedure).

Sodium Azide (preservative and HRP quenching)

Optional humid box for overnight incubation step.

Optional plate shaker for all incubation steps.
6. Preparation of Reagents
6.1 Equilibrate all reagents to room temperature.
6.2 Prepare 1X PBS by diluting 100 mL of 10X PBS in 900 mL
of nanopure water or equivalent. Mix well. Store at room
temperature.
6.3 Prepare 1X Wash Buffer by diluting 1.25 mL of 400X Tween20 in 500 mL of 1X PBS.
Mix well.
Store at room
temperature.
6.4 Immediately prior to use prepare 8% Paraformaldehyde
Solution in 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
6
in a fume hood. Dispose of paraformaldehyde according to
local regulations.
6.5 Prepare 1X Quenching solution by diluting 1.2 mL of 10X
quenching solution in 10.8 mL of nanopure water. Mix well.
Store at room temperature.
6.6 Immediately prior to use prepare 1X Permeabilization
Solution by diluting 0.12 mL of 100X Triton X-100 in 11.88
mL of 1XPBS
6.7 Immediately prior to use prepare 1X Blocking Buffer by
diluting 4.6 mL 10X Blocking Solution and 0.46 mL of 100X
Triton X-100 in 40.94 mL of Nanopure water or equivalent.
6.8 Immediately prior to step 8.10 prepare Development
Solution as follows = 1XFluorescent substrate cocktail + 1X
Hydrogen Peroxide + Fluorescent Substrate Buffer. Discard
any excess after completing the experiment as Hydrogen
Peroxide is unstable in this buffer.
7. Sample Preparation
Note: The protocol below is described for a 96-well plate. If
performing
accordingly.
assay
on
a
384-well
plate,
adjust
volumes
This assay has been optimized for use on
adherent cells. For suspension cells, refer to section 11. Make
certain that the microplate does not dry out at any time before
or during the assay procedure.
7
7.1
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 at
the time of fixation. As an example, MCF7 cells may be
seeded at ~ 25,000 – 50,000 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.
7.2
The attached cells can be treated if desired with a drug of
interest.
7.3
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
100µL 8% Paraformaldehyde to a well with 100 µL media)
or (2) remove culture media from the wells and replace
with 100µL 4% Paraformaldehyde Solution.
7.4
Incubate for 10 minutes at room temperature.
7.5
Remove the Paraformaldehyde Solution from the plate
and wash the plate 3 times briefly with 1X PBS. For each
wash, rinse each well of the plate with 200 µL of 1X PBS.
7.6
Add 100 µL of 1X PBS with 0.02% sodium azide and store
the plate overnight. Sodium azide will preserve the plate
8
for long storage and it will decrease the peroxidase
background normally found on fixed cells. 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
7.7
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 normally found on fixed
cells.
7.8
Wash the plate 3 times with 1X PBS and proceed
immediately to step 8.1.
8. 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 the wells.
Unless
otherwise noted, incubate at room temperature.
During development of this assay we have not observed
problems with edge effects.
However if edge effects are of
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concern, the perimeter wells of the plate can be used as control
wells (primary antibody omitted). Regardless, it is required to
leave at a minimum one well from which the primary antibodies
are excluded to determine background signals of the assay.
8.1
Remove PBS and add 100 µL of Permeabilization Solution
to each well of the plate. Incubate for 30 minutes.
8.2
Remove Permeabilization Solution and add 1X Blocking
buffer to each well of the plate. Incubate for 2 hours.
8.3
Prepare Primary Antibody Cocktail Solution by diluting
100X of each of the primary antibodies into an appropriate
volume of 1X blocking buffer (i.e. 12 mL of 1X Blocking
Buffer + 120 µL of the 100X Anti-AKT1 mouse mAb + 120
µL of the 100X Anti-AKT1 pSer473 Rabbit mAb).
8.4
Remove 1X Blocking buffer and add 100 µL of Primary
Antibody Cocktail Solution to each well.
Incubate
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.
8.5
Remove Primary Antibody 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 step 8.7.
8.6
Prepare 1X Secondary Antibody Cocktail Solution by
diluting 12 µL of 1000X HRP-Labeled anti-rabbit antibody
and 12 µL of 1000X AP-Labeled anti-mouse antibody in 12
mL 1X Blocking buffer.
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8.7
Remove 1X Wash Buffer and add 100 µL 1X Secondary
Antibody Solution to each well of the plate. Incubate 2
hours at room temperature.
8.8
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.
8.9
Wash 2 times with 200 µL per well of 1X PBS.
8.10 Remove PBS and add 100 µL per well of 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
AP substrate = 360 ± 5 nm
spectra
HRP substrate = 555 ± 15 nm
Emission
AP substrate = 449 ± 10 nm
spectra:
HRP substrate = 595 ± 5nm
Time:
up to 60 min
AP signal = between 45
– 60 min
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HRP signal = between
30 – 60 min
Interval:
Shaking:
1 - 5 min
n/a
Shake between
n/a
readings
8.11 Remove the Development solution and wash the plate 3
times with 1X PBS.
8.12 Remove PBS and add 100 µL of Janus Green Stain to
each well of the plate. Incubate plate for 5 minutes at
room temperature.
Note – The RFU signal should be normalized to the Janus
Green staining intensity to account for differences in cell
density.
8.13 Remove the dye and wash the plate 5 times in deionized
water or until excess dye is removed.
8.14 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.
8.15 Measure OD 595 nm using a standard microplate
spectrophotometer.
9. Data Analysis
9.1
Background subtraction. Determine the raw RFU signal
values for each substrate. Subtract the mean background
12
values
from
all
other
RFU
experimental
values
respectively.
9.2
Janus Green normalization of both targets.
Divide the
background subtracted RFU intensities (from 9.1) by the
Janus Green value of the corresponding well. The result
is the “normalized intensity”.
10.
Assay Performance and Specificity
Assay performance was tested using MCF7 cells treated for 30
minutes with 50 nM of purified Insulin from bovine pancreas in 0FMEM media supplemented with pyruvate after overnight serum
starvation. Figure 1 shows performance of the assay on an amine
coated plate. Linearity of raw signal is observed from 12k – 50k per
well.
Figure 1. Dynamic range of assay on Insulin treated MCF7 cells. Cells
were seeded the day before at the specified cell densities, allowed to adhere
and exposed to serum starvation overnight. The next day, cells were treated
13
with 50 nM of Insulin for 30 minutes at 37°C. The signal was obtained using
this kit as described. Total Akt1 with Ex/Em=360/450nm (LEFT) and Akt1
pSer473 with Ex/Em=555/595 nm (RIGHT) are shown as raw signal after
background subtraction (no primary antibody on wells seeded at
corresponding density).
Antibody Specificity and Cross reactivity - Confidence in antibody
specificity is critical to ICE data interpretation.
Therefore, the
antibodies in this kit were tested both in PDGF treated NIH3T3 and
insulin treated MCF7 cells and signal was compared with the
untreated counterparts. Furthermore, specificity was also tested by
fluorescence immunocytochemistry and western blot. The graphs
below show induction of Akt phosphorylation at residue 473 by ICE,
ICC and WB using the antibodies included in this panel.
14
Figure 2. Antibody specificity demonstrated by In Cell ELISA. ICE was
carried out on MCF7 cells seeded at 25,000 cells per well (top) and NIH3T3
cells seeded at 150,000 cells per well (bottom) treated for 30 min with
appropriate compounds after overnight serum starvation. Signal is shown
as a 60 minutes kinetic readout after normalization with janus green. Insulin
treatment increases specific phosphorylation by 25 fold in MCF7 cells,
whereas PDGF treatment increases specific phosphorylation of AKT by 50
fold in NIH3T3 cells.
15
Figure 3. Antibody specificity demonstrated by immunocytochemistry.
ICC was carried out on NIH3T3 cells treated with PDGF (Left) or vehicle
(right) using this kit. Labeling was carried out with a polyclonal antibody
GAR-594 (Akt-1 pSer473) and GAM-488 (Akt-1 total). The PDGF induced
cells (left) show a significant induction of Akt phosphorylation at residue
S473 in comparison to the non-induced control (right).
Figure 4. Antibody specificity demonstrated by WB. Western blot was
run on a 10-20% gradient acrylamide gel. Samples were loaded as follows
from left to right: (1) 50 ng of Human recombinant AKT1 protein (tagged)
Cat# ab62279, (2) 25 µg of non-induced NIH3T3 cell extract and (3) 25 µg of
PDGF induced NIH3T3 cell extract. Membrane Blocking was carried out
with 5% Milk+50 mM Tris+0.05% Tween-20 pH 7.4, primary antibodies were
incubated overnight in 5% BSA+50mM+0.05% Tween-20 pH 7.4 and
16
secondary antibodies were incubated for 2 hours in 5% Milk+50 mM
Tris+0.05% Tween-20 pH 7.4.
Reproducibility
-
ICE
results
provide
accurate
quantitative
measurements of antibody binding and hence cellular antigen
concentrations. The coefficient of the intra-assay variation for this
assay kit on MCF7 cells is typically 6.8% for total and 4.6% for
pSer473. The assay was also found to be highly robust with a mean
Z factor from multiple cell densities (12k – 100k/well) of 0.73 for Akt1 total and 0.81 for Akt-1 pSer473.
11.
Frequently Asked Questions
11.1
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 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.
17
11.2
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.
11.3
A treatment causes cells detachment. Is there a way to
prevent the lost of detaching cells?
Loss of floating cells can be easily prevented by inserting two
centrifugation steps into the protocol: (1) Immediately prior to the
addition of Paraformaldehyde Solution (step 7.3) centrifuge the
microtiter plate at 500x g for 5-10 minutes, (2) Immediately after the
addition of Paraformaldehyde Solution centrifuge the microtiter plate
again at 500x g for 5-10 minutes. Continue in the fixation for a total
of 15 - 20 minutes.
11.4
Can I use suspension cells for ICE?
The In-Cell ELISA can be easily adapted for use with suspension
cells. In this case an amine plate must be used. 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. The
treated suspension cells are then transferred to the amine plate in
100 µLof media per well. The cell seeding density of the amine plate
is cell type-dependent. If necessary, cells can be concentrated by
centrifugation and re-suspended in PBS (preferred) or in media to
18
desired concentration. 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 PBS (preferred) or media. After the cells are transferred to
the amine plate follow immediately the fixation procedure as
described in section 11.3.
Note – With suspension cells, the media should contain no more
than 10 % fetal serum, otherwise efficiency of the suspension cell
cross-linking to the plate may be compromised. When using
suspension cells we recommend not to fix the plate on RPMI media
as this media has been found to increase the assay background on
the AP channel that cannot be quench with the kit’s quenching
buffer.
11.5
I grow my adherent 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.
11.6
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.
11.7
I don’t want to use azide to quench the endogenous HRP
signal on fixed cells. Is there another choice?
19
In the absence of azide, the endogenous HRP signal may be
quenched with a 20 minute treatment of 0.6% H2O2 in PBS.
Following this treatment, cells must be washed thoroughly with PBS
before proceeding with the rest of the experiment.
11.8
Is Janus Green normalization necessary?
Janus Green is a whole-cell stain that is useful to determine if a
decrease in RFU intensity in a well is due to a relevant downregulation 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 (section 9.2).
20
12.
Troubleshooting
Problem
Cause
Endogenous, cellular
alkaline
phosphatase
High AP
background
Growth media
High HRP
background
Endogenous cellular
Peroxidase activity
Solution
Use quenching solution
before the permeabilization
procedure as described in
section 7.7
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% H2O2 in
PBS prior to permeabilizing
the plate
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 section 11
Low Signal
21
Contaminated wash
buffer
Make fresh wash buffer
Edge effects
Do not use the edges of the
plate. Incubate in a humid
box
Variable cell seeding
Plate cells with care and
normalize with Janus Green
Plate is insufficiently
washed
Review the manual for
proper washing. If using a
plate washer, check that all
ports are free from
obstruction
High CV
22
23
24
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26
UK, EU and ROW
Email:
technical@abcam.com
Tel: +44 (0)1223 696000
www.abcam.com
US, Canada and Latin America
Email: us.technical@abcam.com
Tel: 888-77-ABCAM (22226)
www.abcam.com
China and Asia Pacific
Email: hk.technical@abcam.com
Tel: 108008523689 (中國聯通)
www.abcam.cn
Japan
Email: technical@abcam.co.jp
Tel: +81-(0)3-6231-0940
www.abcam.co.jp
Copyright © 2013 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.
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