ab131381 –

GSK3b Total and pSer9

Human Flow Cytometry

Assay Kit

Instructions for Use

For the measurement of GSK3b total and phospho-Ser9 levels in fixed cells. This product is for research use only and is not intended for diagnostic use.

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Table of Contents

1. Introduction 3

2. Assay Summary 5

3. Kit Contents 6

4. Storage and Handling 6

5. Additional Materials Required 7

6. Preparation of Reagents 8

7. Sample Preparation 8

8. Assay Procedure 10

9. Data Analysis 11

10. Assay Performance and Specificity 12

11. Frequently Asked Questions 16

12. Troubleshooting 18

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1. Introduction

Principle: ab131381 is a panel of antibodies that measure the

protein levels of GSK3B total and phosphorylation of GSK3B at

serine residue 9. The assay combines the power of single cell

analysis obtained with flow cytometry and the specificity of

antibody-based immunostaining to quantitate protein and

phosphorylation levels in cultured cells. Cells are harvested and

fixed/permeabilized in suspension, targets of interest are detected

indirectly with highly specific, well-characterized monoclonal

antibodies that are then labeled with fluorescent antibodies.

This flow cytometry panel generates quantitative data with specificity

similar to Western blotting, but with much greater quantitative

precision. This method rapidly fixes the cells in-situ, stabilizing the

in-vivo levels of proteins, and thus essentially eliminates changes

during sample handling, such as with the preparation of protein

extracts.

Background:

Glycogen synthase kinase 3 is a proline directed serine, threonine

kinase originally identified due to its ability to phosphorylate and

inactivate glycogen synthase and later found to be of key importance

in signaling pathways, cell fate determination, energy metabolism,

transcription regulation, neuronal development and body pattern

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formation. There are two isoforms of GSK-3 (alpha and beta) which

show a high degree of homology within their catalytic domains.

Activity of GSK3-B is regulated by phosphorylation of serine 9

(inactivating), phosphorylation of tyrosine 216 (activating) and by

protein complex formation which can activate (Axin, APC, B-catenin)

or inactivate (Frat 1 and 2) the enzyme. GSK3-B regulates by

phosphorylation the activity of numerous metabolic and signaling

proteins such as glycogen synthase, ATP citrate lyase, cyclic-AMP-

dependent protein kinase, acetyl CoA carboxylase, cyclin D1, insulin

receptor substrate-1, pyruvate dehydrogenase amongst others.

Furthermore, it phosphorylates structural cytoskeletal proteins

(MAPs and tau), making it a key component of neuronal structure

and plasticity. GSK3-B is also important for the regulation of

transcription factors that modulate cell survival such as activator

protein-1, cyclic AMP response element binding protein (CREB),

Myc and NFkB.

Altered GSK3B function has been implicated in neuronal

dysfunction. Polymorphisms in the GSK3B gene have been shown

to be associated with an increased risk for Alzheimer disease and

with modifications to the disease risk in Parkinson’s disease due to

its interaction with microtubule associated protein tau (MAPT)

haplotypes. GSK3B has been found to be involved in the

development of psychiatric disorders after the findings that lithium

and valproate, both known to control bipolar disorder, are inhibitors

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of GSK3B. Furthermore the levels of GSK3B have been found

significantly reduced in post-mortem frontal cortex samples from

patients with schizophrenia. GSK3B has also been implicated in

diabetes, cancer and cardiac hypertrophy [PMID: 20331603].

GSK3B has become a key target of pharmacological development in

Alzheimer’s, cancer diabetes, obesity, asthma, immune disorders,

hypertension, AIDS and Glaucoma as indicated by filed patents in

the last decade.

2. Assay Summary

Harvest cells as a single cell suspension.

Fix cells with 4% paraformaldehyde for 15 minutes, pellet and wash.

Permeabilize/Block the cells for 30 minutes at RT then pellet.

Add primary antibodies diluted in 1X incubation buffer, incubate for 1

hour and wash.

Add secondary antibodies diluted in 1X incubation buffer, incubate

for 1 hour, wash and read with flow cytometer.

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3. Kit Contents

Item Quantity

10X Phosphate Buffered Saline (PBS) 100 mL

100X Triton X-100 1.25 mL

Blocking Buffer 10 mL

50X Antibody Cocktail:

Mouse Anti-GSK3B total +

Rabbit Anti-GSK3B pS9 Antibody

220 µL

4. Storage and Handling

Upon receipt, spin down the contents of the antibody cocktail vial.

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

• Flow cytometer

• Microfuge

• Aspirator

• 20% paraformaldehyde

• Goat anti-mouse detection antibodies labeled with

fluorochrome(s) suitable for available flow cytometer

(e.g. Abcam, GAM IgG – H&L DyLight® 488 Cat#ab96879 )

• Goat anti-rabbit detection antibodies labeled with

fluorochrome(s) suitable for available flow cytometer

(e.g. Abcam, GAR IgG – H&L DyLight® 649 Cat# ab96902)

• Nanopure water or equivalent

• Optional for high throughput manipulations: Filter 96-well

plates with pore sizes smaller than the cell line in use

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6. Preparation of Reagents

6.1 Prepare 1X PBS by diluting 100 mL of 10X PBS in 900 mL

Nanopure water or equivalent. Mix well andstore at room

temperature.

6.2 Immediately prior to use prepare

2X Blocking-Permeabilization Buffer (2X Triton X-100,

20% Blocking Buffer in PBS.) Discard any excess.

6.3 Immediately prior to use prepare 1X Incubation Buffer

(10% Blocking Solution in PBS). Any excess should be

stored at -20˚C.

7. Sample Preparation

7.1 Cell culture and treatment conditions are dictated by the

experiment at hand. As a general guideline, it is advisable

to analyze at least 10,000 events (cells) on the flow

cytometer per sample/data point. Therefore at least four

to ten times that many cells should be collected per data

point to ensure sufficient material at the end of the

staining.

7.2 For suspension cells, generate a single cell solution by

pipetting up and down.

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7.3 For adherent cells, fully dissociate cells (e.g. trypsin) into

single cell suspension. Passaging the cell line the day

before the experiment onto a fresh culture plate may help

improve single cell dissociation on the day of the

experiment.

7.4 Maintain cells resuspended in the culture treatment media,

at approximately 1x106 cells per mL.

7.5 Overlay 20% paraformaldehyde on the cell suspension so

that the final concentration is 4%, gently mix by inverting

the tube and incubate at room temperature for 15 minutes.

7.6 Pellet cells at 350 - 500 x g for 5 minutes (depending on

cell size) and decant supernatant.

Note: paraformaldehyde is toxic: handle with care and

dispose of according to local requirements

7.7 Dislodge the pellet by gently tapping the bottom of the

tube and resuspend the cells in 1X PBS (1x105 per

0.05 mL) and aliquot into assay vials.

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8. Assay Procedure

Note: Provided reagents are sufficient for 100 tests in a 100 µL

volume or 200 tests in a 50 µL volume. Always include negative

controls as follows (1) – primary/+secondary and (2) – primary/-

secondary. After every centrifugation step, aspirate the

supernatant leaving 50 µL volume inside the assay vial and

gently tap the bottom of the tube to dislodge the pellet before

continuing to the next step.

8.1 Overlay an equal volume of 2X Blocking-Permeabilization

buffer into each assay vial (e.g. 50 µL of cell suspension +

50 µL of 2X Blocking-Permeabilization buffer) and

incubate for 30 minutes at room temperature.

8.2 Pellet cells at 350 - 500 x g for 5 minutes (depending on

cell size) and aspirate supernatant.

8.3 Prepare 50 µL (or 100 µL if using 100 tests) per assay

tube of 2X Primary Antibody Cocktail Solution in

1X Incubation Buffer (1:25 dilution of 50X Antibody

Cocktail) in order to overlay the 50 µL cell suspension (or

100 µL if using 100 tests) for a final 1X Antibody Cocktail

solution. Incubate at room temperature for at least 1 hour.

8.4 Pellet cells at 350 - 500 x g for 5 minutes (depending on

cell size) and aspirate supernatant.

8.5 Wash twice by centrifugation with 1 mL of 1X Incubation

Buffer per assay tube.

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8.6 Prepare 100 µL per assay tube of a Goat Anti-Mouse and

Goat Anti-Rabbit fluorochrome labeled cocktail solution in

1X Incubation Buffer per assay tube.

8.7 Add 100 µL of detector solution per assay tube. Incubate

for 1 hour at room temperature in the dark.

8.8 Pellet cells at 350 – 500 x g for 5 minutes (depending on

cell size) and aspirate supernatant.

8.9 Wash twice by centrifugation with 1 mL of 1X Incubation

Buffer per assay tube.

8.10 Add 100 µL of 1X PBS to each assay tube before reading

on the flow cytometer.

9. Data Analysis

Specific methods depend on the available flow cytometer. It is

important to appropriately establish forward and side scatter gates to

exclude debris and cellular aggregates from analysis. Certain

treatments may generate subpopulations of cells that are apparent

from the forward/side scatter plots. Under these circumstances it is

recommended to adequately gate the subpopulation of interest

before capturing events. If the histogram does not generate a

perfect normal distribution, use the median measurement to prevent

artifacts from skewing the data.

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10. Assay Performance and Specificity

Specificity

Assay specificity was demonstrated by using Jurkat cells grown in

RPMI media with 10% FCS and treated for 60 minutes with 100 nM

Calyculin or DMSO (vehicle control). Figure 1 shows flow cytometry

results of this cell-culture model.

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Figure 1. Antibody specificity demonstrated by Flow cytometry. Non-

induced Jurkat cells (red) and calyculin induced (blue) were targeted with

the antibody cocktail against GSK3B total and GSK3B (pSer9).

Background fluorescence (pink) was determined with a no primary-antibody

control. Primary antibodies were labeled with 1:500 dilution of GAM IgG –

H&L DyLight® 488 Cat# ab96879 and 1:500 dilution of GAR IgG – H&L

DyLight® 649 Cat# ab96902 respectively. Signal intensity was measured in

FL-1 (GSK3B total) and FL-4 (GSK3B pSer9). After background subtraction,

the calyculin induced cell line shows a 6 fold increase in the levels of

phosphorylated GSK3B in comparison to a 1.2 decrease in the levels of

GSK3B protein.

Confidence in antibody specificity is critical to Flow data

interpretation. Therefore, the antibodies in this kit were also tested

for specificity by fluorescence immunocytochemistry and western

blot using the same aforementioned cell-culture model. The images

below show induction of GSK3B phosphorylation at residue 9 both

by ICC and WB using the antibodies included in this panel.

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Figure 2. Antibody specificity demonstrated by immunocytochemistry.

ICC was carried out on HeLa cells treated with calyculin (left) or vehicle

(right) with Rabbit anti-GSK3B pSer9 and Mouse anti-GSK3B using all buffer

reagents as supplied in this kit. Labeling was carried out with 1:500 dilution

of GAR IgG - H&L DyLight® 594 Cat# ab96897 and 1:500 dilution of GAM

IgG – H&L DyLight® 488 Cat# ab96879 respectively. The calyculin induced

cells (left) show a significant induction of GSK3B phosphorylation at residue

S9 in comparison to the non-induced control (right).

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Figure 3. Validation of antibodies by WB. Western blot was run on a

4-20% gradient acrylamide gel. Samples were loaded as follows from left to

right: (1)50 ng GSK3B protein (ab63193), (2) 40 µg of Jurkat cells treated

with DMSO, (3) 40 µg of Jurkat cells treated with 100nM calyculin, (4) 40 µg

of HeLa cells treated with DMSO, (5) 40 µg of HeLa cells treated with

100 nM calyculin. Membrane blocking and incubation of primary and

secondary antibodies was carried out with 1X Blocking Buffer (ab126587) for

anti-GSK3B and anti-GSK3B pSer9. Actin was targeted with ab8224

following recommendations on product sheet.

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11. Frequently Asked Questions

11.1 The cells I am working with are extremely fragile and they

lyse during sample processing. How can I avoid this?

For extremely fragile cells we recommend to fix and

block/permeabilize the cells as suggested in this booklet

followed by a short staining procedure as follows:

11.1.1. Overlay 100 µL of 2X primary antibody cocktail

solution on cells suspension (in

Blocking/permeabilization buffer) and incubate for 1

hour at room temperature.

11.1.2. Centrifuge at 400g for 4 minutes and remove

supernatant. Do not wash.

11.1.3. Add 100 µL of 1x secondary antibody cocktail

solution and incubate for 1 hour at room

temperature in the dark.

11.1.4. Centrifuge at 400g for 4 minutes, remove

supernatant and wash once with 500 µL of

incubation buffer.

11.2 I grow my cells in 10% FBS, will this interfere with the cell

fixation?

Culture media containing up to 10% fetal serum does not

interfere with the cell fixation.

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11.3 How do I measure the assay background?

It is essential to omit primary antibody in at least one assay

vial to provide a background signal for the experiment which

can be subtracted from all measured data.

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12. Troubleshooting

Problem Cause Solution

Low Signal

Signal not correctly compensated

Check positive single color control is set up correctly

on flow cytometer and gated/compensated

correctly to capture all events

Insufficient secondary antibody

Increase concentration of antibody

Lasers not aligned Run flow check beads and

adjust alignment if necessary

Secondary antibody is not compatible

with primaries

Use secondary goat antibodies raised against mouse and rabbit for this

kit

High side scatter back-

ground

Cells lysed

Ideally samples should be freshly prepared. Do not

vortex or shake the sample at any stage. Do not exceed 500 x g for

centrifugation Bacterial

contamination Ensure sample is not

contaminated

Low event rate

Low number of cells Run 1x106 cells/mL

Cells clumped Ensure a single cell

suspension. Sieve clumps (30 µL nylon mesh)

High event rate

High number of cells/mL

Dilute between 1x105

cells/mL and 1x106

cells/mL

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