Store at -20°C

Akt Kinase Assay Kit

(Nonradioactive)
31 Kit 40 assays
n Orders n 877-616-CELL (2355)
[email protected]
#9840

Support n 877-678-TECH (8324)

[email protected]
Web n www.cellsignal.com
rev. 05/02/16

For Research Use Only. Not For Use In Diagnostic Procedures.

Description: Nonradioactive Akt Kinase Assay Kit provides

Products Included Product # Kit Quantity
all the reagents necessary to measure Akt kinase activity in
the cell. Immobilized Phospho-Akt (Ser473) (D9E) Rabbit Immobilized Phospho-Akt (Ser473) (D9E) Rabbit mAb (Bead Conjugate) 4070 1 x 800 µl (40 immunoprecipitations)
mAb is used to immunoprecipitate Akt from cell extracts.
Phospho-GSK-3α/β (Ser21/9) (37F11) Rabbit mAb (GSK-3α Preferred) 9327 1 x 100 µl (10 Western mini-blots)
Then, an in vitro kinase assay is performed using GSK-3
Fusion Protein as a substrate. Phosphorylation of GSK-3 is GSK 3 Fusion Protein 9237 1 x 40 µg
measured by Western blotting, using Phospho-GSK-3a/b Kinase Buffer (10X) 9802 1 x 15 ml
(Ser21/9) Antibody.
Cell Lysis Buffer (10X) 9803 1 x 15 ml
Species Cross Reactivity: H, M, R
ATP (10 mM) 9804 1 x 50 µl
kDa Anti-rabbit IgG, HRP-linked Antibody 7074 1 x 100 µl
200 Anti-biotin, HRP-linked Antibody 7075 1 x 100 µl
140
20X LumiGLO Reagent and 20X Peroxide
®
7003 5 ml each
100
80 Biotinylated Protein Ladder Detection Pack 7727 1 x 100 µl

60
Akt Kinase Assay Kit Overview
50

40 Step 1: Selective IP of Akt using Immobilized Akt Antibody.

30 Phospho-GSK-
3α/β (Ser21/9)

20

+ – Akt
0.5 0.5 µg of GSK-3α/β
fusion protein used

AKT Kinase activity of PDGF-treated NIH/3T3 cell extracts was

Immobilized
analyzed by IP/Kinase assay. Cell extracts (200 μl) were incu- Akt Ab
Akt
bated overnight with Immobilized Phospho-Akt (Ser473) (D9E)
Rabbit mAb #4070. After extensive washing the kinase reaction cell extract cell extract
was performed in the presence of 200 μM of cold ATP and 1 μg
of GSK-substrate. Phosphorylation of GSK-3 was measured by a) Add immobilized Akt Antibody b) IP Akt from cell extracts using Immobilized Akt Antibody.
Western blot using Phospho-GSK-3 a/β (Ser21/9) Antibody
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

#9327. Step 2: Incubate IP pellets in Kinase Buffer containing GSK-3 fusion protein and cold ATP.

Molecular Weight: 27 kDa

P P P
Kit Components: Thr308 Ser473 Ser21/9 Ser21/9
Immobilized Phospho-Akt (Ser473) (D9E) Rabbit
+ATP
mAb (Bead Conjugate): Phospho-Akt (Ser473) (D9E) Akt +
Rabbit mAb (Bead Conjugate) immunoprecipitates endog- IP
enous levels of Akt only when phosphorylated at Ser473. (Akt) (Active)
Store at -20ºC. Do not aliquot the antibody.
Step 3: Detect GSK-3 phosphorylation using phospho-GSK-3a/b (Ser21/9) antibodies by Western blotting
Phospho-GSK-3α/β (Ser21/9) (37F11) Rabbit mAb and chemiluminescent detection.
© 2016 Cell Signaling Technology, Inc.

(GSK-3α Preferred):Phospho-GSK-3a/b (Ser21/9)

(37F11) Rabbit mAb (GSK-3a Preferred) preferentially
detects endogenous levels of GSK-3a when phosphorylated
at Ser21 and also detects GSK-3b when phosphorylated at
Ser9. Store at –20°C. Do not aliquot the antibody.

U.S. Patent No. 5,675,063

page 1 of 3

Tween® is a registered trademark of ICI Americas, Inc.

Species Cross-Reactivity Key: H—human M—mouse R—rat Hm—hamster Mk—monkey Mi—mink C—chicken X—Xenopus Z—zebra fish B—bovine All—all species expected
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
 GSK-3 Fusion Protein: GSK-3α/β crosstide corre- Selected Application References: Companion Products:
sponding to residues surrounding GSK-3α/β (Ser21/9) Hisamoto, K. et al. (2000) Estrogen induces the Akt-de- Wortmannin #9951
(CGPKGPGRRGRRRTSSFAEG) is expressed as a GST fusion pendent activation of endothelial nitric oxide synthase in
vascular endothelial cells. J. Biol. Chem. 276, 3459–3467. LY294002 (PI3 Kinase Inhibitor) #9901
protein.
Applications: Kinase Assay, W. PhosphoPlus® Akt (Ser473) Antibody Kit #9270
10X Kinase Buffer: 1X concentration: 25 mM Tris (pH 7.5),
5 mM b-Glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, Deregibus, M.C. et al. (2003) CD40-dependent activation of Phospho-Akt (Ser473) Antibody #9271
10 mM MgCl2. phosphatidylinositol 3-kinase/Akt pathway mediates endo- Phospho-Akt (Ser473) (587F11) Mouse mAb #4051
thelial cell survival and in vitro angiogensis. J. Biol. Chem.
10X Cell Lysis Buffer: 1X concentration: 20 mM Tris Phospho-Akt (Thr308) Antibody #9275
278, 18008–18014. Applications: kinase assay, W.
(pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Jost, M. et al. (2001) Epidermal growth factor receptor-de- Phospho-Bad (Ser112) Antibody #9291
Triton, 2.5 mM sodium pyrophosphate, 1 mM b-glycero-
phosphate, 1 mM Na3VO4, 1 µg/ml Leupeptin. pendent control of keratinocyte survival and Bcl-xL expres- Phospho-Bad (Ser136) Antibody #9295
sion through a MEK-dependent pathway. J. Biol. Chem.
10 mM ATP (50 µl): Adenosine-5’ triphosphate (ATP) Phospho-GSK-3β (Ser9) Antibody #9336
276, 6320–6326. Applications: Kinase Assay, W.
supplied as a 10 mM solution in sterile, doubly distilled Phospho-Tuberin/TSC2 (Thr1462) Antibody #3611
water as a disodium salt. Thimmaiah, K.N. et al. (2003) Insulin-like growth factor
I-mediated protection from rapamycin-induced apoptosis is Phospho-Tuberin/TSC2 (Tyr1571) Antibody #3614
Phototope-HRP Western Detection Kit: The Phototope
independent of Ras-Erk1-Erk2 and phosphatidylinositol 3’- GSK-3β (27C10) Rabbit mAb #9315
Western Detection System contains sufficient reagents for
kinase-Akt signaling pathways. Cancer Res. 63, 364–374.
the chemiluminescent detection of rabbit antibodies on 10 Akt Antibody #9272
Applications: Kinase Assay, W.
(10 cm x 10 cm) Western blots. It includes a secondary anti- Prestained Protein Marker, Broad Range (Premixed Format)
rabbit antibody conjugated to horseradish peroxidase, anti- Kim, S. et al. (2004) Akt activation in platelets depends on
#7720
biotin antibody conjugated to horseradish peroxidase for Gi signaling pathways. J. Biol. Chem. 279, 4186–4195.
the detection of the biotinylated protein ladder (included), Applications: Kinase Assay, W. Biotinylated Protein Ladder Detection Pack #7727
LumiGLO® chemiluminescent reagent, and peroxide. Background References:
Cavin, L.G. et al. (2005) Transforming growth factor-a
Background: Akt, also referred to as PKB or Rac, plays a inhibits the intrinsic pathway of c-Myc-induced apoptosis (1) F ranke, T.F. et al. (1997) Cell 88, 435–7.
critical role in controlling survival and apoptosis (1-3). This through activation of nuclear factor-kB in murine hepatocel-
(2) B urgering, B.M. and Coffer, P.J. (1995) Nature 376,
protein kinase is activated by insulin and various growth lular carcinomas. Mol. Cancer Res. 3, 403–412. Applica-
599–602.
and survival factors to function in a wortmannin-sensitive tions: Kinase Assay, W.
pathway involving PI3 kinase (2,3). Akt is activated by (3) F ranke, T.F. et al. (1995) Cell 81, 727–36.
Hu, H. et al. (2005) PKB/AKT and ERK regulation of cas-
phospholipid binding and activation loop phosphorylation (4) A lessi, D.R. et al. (1996) EMBO J 15, 6541–51.
pase-mediated apoptosis by methylseleninic acid in LNCaP
at Thr308 by PDK1 (4) and by phosphorylation within the
prostate cancer cells. Carcinogenesis 26, 1374–1381. (5) S arbassov, D.D. et al. (2005) Science 307, 1098–101.
carboxy terminus at Ser473. The previously elusive PDK2
Applications: Kinase Assay, W.
responsible for phosphorylation of Akt at Ser473 has been (6) J acinto, E. et al. (2006) Cell 127, 125–37.
identified as mammalian target of rapamycin (mTor) in a
(7) C
 ardone, M.H. et al. (1998) Science 282, 1318–21.
rapamycin-insensitive complex with rictor and Sin1 (5,6).
Akt promotes cell survival by inhibiting apoptosis by (8) B runet, A. et al. (1999) Cell 96, 857–68.
phosphorylating and inactivating several targets, including (9) Z immermann, S. and Moelling, K. (1999) Science 286,
Bad (7), forkhead transcription factors (8), c-Raf (9) and 1741–4.
caspase-9. PTEN phosphatase is a major negative regulator
of the PI3 kinase/Akt signaling pathway (10). LY294002 is a (10) C
 antley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci
specific PI3 kinase inhibitor (11). USA 96, 4240–5.
(11) V lahos, C.J. et al. (1994) J Biol Chem 269, 5241–8.
Another essential Akt function is the regulation of glycogen
synthesis through phosphorylation and inactivation of (12) H
 ajduch, E. et al. (2001) FEBS Lett 492, 199–203.
GSK-3α and β (12,13). Akt may also play a role in insulin (13) C
 ross, D.A. et al. (1995) Nature 378, 785–9.
stimulation of glucose transport (12).
(14) D
 iehl, J.A. et al. (1998) Genes Dev 12, 3499–511.
In addition to its role in survival and glycogen synthesis, Akt
is involved in cell cycle regulation by preventing GSK-3β (15) G
 esbert, F. et al. (2000) J Biol Chem 275, 39223–30.
mediated phosphorylation and degradation of cyclin D1 (14) (16) Z hou, B.P. et al. (2001) Nat Cell Biol 3, 245–52.
and by negatively regulating the cyclin dependent kinase
(17) N
 avé, B.T. et al. (1999) Biochem J 344 Pt 2, 427–31.
inhibitors p27 Kip (15) and p21 Waf1 (16). Akt also plays a
critical role in cell growth by directly phosphorylating mTOR (18) Inoki, K. et al. (2002) Nat Cell Biol 4, 648–57.
in a rapamycin-sensitive complex containing raptor (17). (19) M
 anning, B.D. et al. (2002) Mol Cell 10, 151–62.
More importantly, Akt phosphorylates and inactivates tu-
berin (TSC2), an inhibitor of mTOR within the mTOR-raptor
complex (18). Inhibition of mTOR stops the protein synthesis
machinery due to inactivation of its effector, p70 S6 kinase
and activation of the eukaryotic initiation factor 4E binding
protein 1 (4E-EP1), an inhibitor of translation (18,19).
® 2014 Cell Signaling Technology, Inc.

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Orders n 877-616-CELL (2355) [email protected] Support n 877-678-TECH (8324) [email protected] Web n www.cellsignal.com
 #9840
Nonradioactive IP-Kinase Assay Protocol

A Solutions and Reagents D Kinase Assay

1. Note: Prepare solutions with Milli-Q or equivalently purified water. 1. Microcentrifuge cell lysate/immobilized antibody at 14,000 x G for 30 seconds
2. 1X Cell Lysis Buffer: May be stored at 4°C for short-term use at 4°C. Wash pellet two times with 500 µL of 1X Cell Lysis Buffer. Keep on ice
(1–2 weeks). Note: Supplied 10X Cell Lysis Buffer should be vortexed during washes.
before being used to make 1X solution. 2. Wash pellet twice with 500 µL of 1X Kinase Buffer. Keep on ice.
3. 1X Kinase Buffer: Store at –20°C. May be stored at 4°C for short-term 3. Suspend pellet in 50 µL of 1X Kinase Buffer supplemented with 1 µl of 10 mM
use (1–2 weeks). ATP and appropriate quantity of kinase substrate (1µl).
4. GSK-3 Fusion Protein: Concentration = 0.5 mg/ml. Use 0.5 µg/assay. 4. Incubate for 30 minutes at 30°C.
5. 10 mM ATP Adenosine-5’ triphosphate (ATP) supplied as a 10 mM solu- 5. Terminate reaction with 25 µL 3X SDS Sample Buffer. Vortex, then microcentri-
tion in sterile, doubly distilled water as a disodium salt. fuge for 30 seconds at 14,000 x G.
6.* Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol (pH=8.5).
7.* 3X SDS Sample Buffer: 187.5 mM Tris-HCl (pH 6.8 at 25°C), 6% w/v E Western Immunoblotting
sodium dodecyl sulfate (SDS), 30% glycerol, 150 mM dithiothreitol (DTT), 1. Heat the sample to 95–100°C for 2–5 minutes.
0.03% w/v bromophenol blue. For 100 mL, use 2.27 g Tris-HCl, 6g SDS, 2. Load 5-15 µl of sample per well sample on SDS-PAGE gel.
30 mL glycerol and 30mg w/v bromophenol blue or bromophenol blue 3. Note: CST recommends loading prestained molecular weight markers (#7720,
dye. Store at –20°C. Add DTT fresh just before use. 10 µL/lane) to verify electrotransfer and biotinylated protein marker (#7727,
8.*  10X Tris-Buffered Saline with Tween®20 (TBS/T): 0.2 M Tris base, 1.36 10 µL/lane) to estimate molecular weights.
M NaCl, 1.0% Tween®20. To prepare 1 liter, dissolve 24.2 g Tris, 80 g NaCl 4. Run SDS-page and electrotransfer to nitrocellulose or PVDF membrane.
in dH2O and adjust pH to 7.6 with HCl. Store at room temperature. 5. Note: Volumes for all the following steps are for 10 cm x 10 cm membrane; for
9.* Blocking Buffer: 1X TBS/T with 5% w/v nonfat dry milk. For 150 mL, dissolve different sized membranes, adjust volumes accordingly.
7.5g nonfat dry milk in 15 mL 10X TBS/T and 135 mL dH2O. Mix well. Prepare 6. (Optional) After transfer, wash nitrocellulose membrane with 25 mL TBST for 5
freshly for each experiment. minutes at room temperature.
10.* Wash Buffer: 1X TBS, 0.1% Tween®20 (TBS/T). Store at room temperature. 7. Incubate membrane in 10 mL Blocking Buffer for 1-2 hours at room
11.* Primary Antibody Dilution Buffer: 1X TBS/T with 5% BSA. temperature.
12.  Phototope-HRP Western Blot Detection System #7071: Includes 8. Wash three times for 5 minutes each with 15 mL Wash Buffer.
biotinylated protein marker, secondary anti-rabbit (#7074) antibody conjugated 9. Incubate membrane and Phospho-GSK-3a/b (Ser21/9) Antibody (1:1000 dilu-
to horseradish peroxidase (HRP), Anti-biotin, HRP-linked (D5A7) Rabbit mAb tion) in 10 mL Primary Antibody Dilution Buffer with gentle agitation overnight
(#5571), 20X LumiGLO® chemiluminescent reagent and 20X peroxide (#7003). at 4°C.
13. LumiGLO® Substrate #7003: 0.5 mL 20X LumiGLO, 0.5 mL 20X peroxide 10. Wash three times for 5 minutes each with 15 mL Wash Buffer.
and 9.0 mL Milli-Q water. 11. Incubate membrane with HRP-conjugated secondary antibody (1:2000) and
HRP-conjugated anti-biotin antibody (1:1000) to detect biotinylated protein
B Preparing Cell Lysates markers in 10 mL of Blocking Buffer with gentle agitation for 1 hour at room
1. Aspirate media. Treat cells by adding fresh media containing regulator temperature.
for desired time. 12. Wash three times for 5 minutes each with 15 mL Wash Buffer.
2. To harvest cells under nondenaturing conditions, remove media and rinse cells 13. Incubate membrane with 10 mL LumiGLO Substrate with gentle
once with ice-cold PBS. agitation for 1 minute at room temperature.
3. Remove PBS and add 0.5 mL ice-cold 1X Cell Lysis Buffer plus 14. Drain membrane of excess LumiGLO® Substrate (but do not let dry),
1 mM phenylmethylsulfonyl fluoride (PMSF) to each plate (10 cm2) and incubate wrap in plastic wrap and expose to X-ray film. An initial 10-second
the plate on ice for 5 minutes. exposure should indicate the proper exposure time.
4. Scrape cells off the plate and transfer to an appropriate tube. Keep on ice. 15. Note: Due to the kinetics of the detection reaction, signal is most intense
5. Sonicate lysates on ice. immediately following LumiGLO® incubation and declines over the following 2
6. Microcentrifuge for 10 minutes at 4°C and transfer the supernatant hours. LumiGLO® Substrate can be further diluted if signal response is too fast.
to a new tube. The supernatant is the cell lysate. Store at –80°C.

C IP with Immobilized Antibodies

1. Instructions for use: Prior to use, put tube on ice for 5 minutes to lower viscosity
of buffer. Then Beads should be resuspended to a 50% slurry be inversion or
gentle vortexing.
2. For immunoprecipitations with immobilized Akt primary antibody:
Add 20 µl of immobilized antibody bead slurry to 200 µl cell lysate. Incubate
with gentle rocking overnight at 4°C.
® 2014 Cell Signaling Technology, Inc.

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Orders n 877-616-CELL (2355) [email protected] Support n 877-678-TECH (8324) [email protected] Web n www.cellsignal.com