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ELISA Antibody Pair

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. Wash Buffer: 1X PBS/0.05% Tween® 20, (20X PBST #9809).
  3. Blocking Buffer: 1X PBS/0.05% Tween® 20, 1% BSA.
  4. 1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).

    Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.

  5. Bovine Serum Albumin (BSA): (#9998).
  6. TMB Substrate: (#7004).
  7. STOP Solution: (#7002)

    NOTE: Reagents should be made fresh daily.

B. Preparing Cell Lysates

For adherent cells

  1. Aspirate media when the culture reaches 80–90% confluence. Treat cells by adding fresh media containing regulator for desired time.
  2. Remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X Cell Lysis Buffer plus 1 mM PMSF to each plate (10 cm diameter) and incubate the plate on ice for 5 min.
  4. Scrape cells off the plate and transfer to an appropriate tube. Keep on ice.
  5. Sonicate lysates on ice.
  6. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at -80°C in single-use aliquots.

For suspension cells

  1. Remove media by low speed centrifugation (~1,200 rpm) when the culture reaches 0.5–1.0 x 106 viable cells/ml. Treat cells by adding fresh media containing regulator for desired time.
  2. Collect cells by low speed centrifugation (~1,200 rpm) and wash once with 5–10 ml ice-cold 1X PBS.
  3. Cells harvested from 50 ml of growth media can be lysed in 2.0 ml of 1X cell lysis buffer plus 1 mM PMSF.
  4. Sonicate lysates on ice.
  5. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at -80°C in single-use aliquots.

C. Coating Procedure

  1. Rinse microplate with 200 µl of dH2O, discard liquid. Blot on paper towel to make sure wells are dry.
  2. Dilute capture antibody 1:100 in 1X PBS. For a single 96 well plate, add 100 µl of capture antibody stock to 9.9 ml 1X PBS. Mix well and add 100 µl/well. Cover plate and incubate overnight at 4°C (17–20 hr).
  3. After overnight coating, gently uncover plate and wash wells:
    1. Discard plate contents into a receptacle.
    2. Wash four times with wash buffer, 200 µl each time per well. For each wash, strike plates on fresh paper towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time.
    3. Clean the underside of all wells with a lint-free tissue.
  4. Block plates. Add 150 µl of blocking buffer/well, cover plate, and incubate at 37°C for 2 hr.
  5. After blocking, wash plate (Section C, Step 3). Plate is ready to use.

D. Test Procedure

  1. Lysates can be used undiluted or diluted in blocking buffer. 100 µl of lysate is added per well. Cover plate and incubate at 37°C for 2 hr.
  2. Wash plate (Section C, Step 3).
  3. Dilute detection antibody 1:100 in blocking buffer. For a single 96 well plate, add 100 µl of detection antibody Stock to 9.9 ml of blocking buffer. Mix well and add 100 µl/well. Cover plate and incubate at 37°C for 1 hr.
  4. Wash plate (Section C, Step 3).
  5. Secondary antibody, either streptavidin anti-mouse or anti-rabbit-HRP, is diluted 1:1000 in blocking buffer. For a single 96 well plate, add 10 µl of secondary antibody stock to 9.99 ml of blocking buffer. Mix well and add 100 µl/well. Cover and incubate at 37°C for 30 min.
  6. Wash plate (Section C, Step 3).
  7. Add 100 µl of TMB substrate per well. Cover and incubate at 37°C for 10 min.
  8. Add 100 µl of STOP solution per well. Shake gently for a few seconds.
  9. Read plate on a microplate reader at absorbance 450 nm.
    1. Visual Determination: Read within 30 min after adding STOP solution.
    2. Spectrophotometric Determination: Wipe underside of wells with a lint-free tissue. Read absorbance at 450 nm within 30 min after adding STOP solution.

posted January 2008

revised Sepetember 2013

protocol id: 20

Product Includes Volume Cap Color
Akt Capture Ab (100X) 0.4 ml Pink
Akt1 Detection Ab (100X) 0.4 ml Blue
Anti-mouse IgG, HRP-linked Antibody (1000X) 0.04 ml Yellow

Product Description

CST's PathScan® Total Akt1 Sandwich ELISA Antibody Pair is being offered as an economical alternative to our PathScan® Total Akt1 Sandwich ELISA Kit #7170. Capture and Detection antibodies (100X stocks) and HRP-conjugated secondary antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The Akt Rabbit Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by Akt1 Mouse Detection Antibody and HRP-conjugated Anti-Mouse IgG. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of Total Akt1 protein.

Antibodies in kit are custom formulations specific to kit.


Product Usage Information

Antibody pairs have been optimized using recommended buffers, reagents, plates and the included protocol. Solutions should be made fresh daily.


Storage: Capture and detection antibodies are stored at 4°C. HRP-linked secondary reagent is stored at -20°C.

Specificity / Sensitivity

For Antibody Pair specificity and sensitivity, please refer to the corresponding PathScan® Sandwich ELISA Kit. Note: This antibody pair detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.


Species Reactivity: Human, Mouse

Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 (15) and p21 Waf1/Cip1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18,19).


1.  Franke, T.F. et al. (1997) Cell 88, 435-7.

2.  Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.

3.  Franke, T.F. et al. (1995) Cell 81, 727-36.

4.  Cross, D.A. et al. (1995) Nature 378, 785-9.

5.  Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.

6.  Sarbassov, D.D. et al. (2005) Science 307, 1098-101.

7.  Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511.

8.  Jacinto, E. et al. (2006) Cell 127, 125-37.

9.  Cardone, M.H. et al. (1998) Science 282, 1318-21.

10.  Brunet, A. et al. (1999) Cell 96, 857-68.

11.  Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.

12.  Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.

13.  Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.

14.  Hajduch, E. et al. (2001) FEBS Lett 492, 199-203.

15.  Gesbert, F. et al. (2000) J Biol Chem 275, 39223-30.

16.  Zhou, B.P. et al. (2001) Nat Cell Biol 3, 245-52.

17.  Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.

18.  Inoki, K. et al. (2002) Nat Cell Biol 4, 648-57.

19.  Manning, B.D. et al. (2002) Mol Cell 10, 151-62.


Entrez-Gene Id 207
Swiss-Prot Acc. P31749

Protein Specific References

Germack R and Dickenson JM (2000) Br J Pharmacol 130, 867–74

Wick MJ et al. (2000) J Biol Chem 275, 40400–6

Rane MJ et al. (2001) J Biol Chem 276, 3517–23

Guizzetti M and Costa LG (2001) Neuroreport 12, 1639–42

Brognard J et al. (2001) Cancer Res 61, 3986–97

Maira SM et al. (2001) Science 294, 374–80

Schönherr E et al. (2001) J Biol Chem 276, 40687–92

Hill MM et al. (2001) J Biol Chem 276, 25643–6

Dhawan P et al. (2002) Cancer Res 62, 7335–42

Conus NM et al. (2002) J Biol Chem 277, 38021–8

Sano H et al. (2002) J Biol Chem 277, 19439–47

Egawa K et al. (2002) J Biol Chem 277, 38863–9

Kisseleva MV et al. (2002) J Biol Chem 277, 6266–72

Barry FA and Gibbins JM (2002) J Biol Chem 277, 12874–8

Ikonomov OC et al. (2002) Endocrinology 143, 4742–54

Rani MR et al. (2002) J Biol Chem 277, 38456–61

Ho R et al. (2002) Cancer Res 62, 6462–6

Wan X and Helman LJ (2003) Oncogene 22, 8205–11

Fukuda T et al. (2003) J Biol Chem 278, 51324–33

Kim HH et al. (2003) FASEB J 17, 2163–5

Min YH et al. (2004) Cancer Res 64, 5225–31

Tazzari PL et al. (2004) Br J Haematol 126, 675–81

Matsuzaki H et al. (2004) Biochemistry 43, 4284–93

Wolfrum S et al. (2004) Arterioscler Thromb Vasc Biol 24, 1842–7

Kaneko Y et al. (2004) J Cell Sci 117, 407–15

Esfandiarei M et al. (2004) J Virol 78, 4289–98

Baudhuin LM et al. (2004) FASEB J 18, 341–3

Dietze EC et al. (2004) Oncogene 23, 3851–62

Wu T et al. (2004) Mol Cancer Ther 3, 299–307

Honjo S et al. (2005) DNA Cell Biol 24, 141–7

Karlsson HK et al. (2005) Diabetes 54, 1459–67

Viniegra JG et al. (2005) J Biol Chem 280, 4029–36

Le XF et al. (2005) J Biol Chem 280, 2092–104

Smith E and Frenkel B (2005) J Biol Chem 280, 2388–94

Edwards LA et al. (2005) Oncogene 24, 3596–605

Karlsson HK et al. (2005) Diabetes 54, 1692–7

Kippenberger S et al. (2005) J Biol Chem 280, 3060–7

Jung HS et al. (2005) Mol Endocrinol 19, 2748–59

Khundmiri SJ et al. (2006) Am J Physiol Cell Physiol 291, C1247–57

Hers I and (2007) Blood 110, 4243–52

Ananthanarayanan B et al. (2007) J Biol Chem 282, 36634–41

Zunder ER et al. (2008) Cancer Cell 14, 180–92

Grenegård M et al. (2008) J Biol Chem 283, 18493–504

Abubaker J et al. (2009) Mol Cancer 8, 51

Chen PL and Easton AS (2011) Curr Neurovasc Res 8, 14–24

Van Aller GS et al. (2011) Biochem Biophys Res Commun 406, 194–9

Uesugi A et al. (2011) Cancer Res 71, 5765–78

Ou YH et al. (2011) Mol Cell 41, 458–70

Wang S et al. (2012) PLoS One 7, e37427

Glidden EJ et al. (2012) J Biol Chem 287, 581–8

Shih MC et al. (2012) Oncogene 31, 2389–400

Misra UK and Pizzo SV (2012) J Cell Biochem 113, 1488–500


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
PathScan® is a trademark of Cell Signaling Technology, Inc.