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54336
FastScan™ Phospho-Akt1 (Ser473) ELISA Kit
ELISA Kits

FastScan™ Phospho-Akt1 (Ser473) ELISA Kit #54336

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Figure 1. Treatment of MCF7 cells with IGF-1 stimulates phosphorylation of Akt1 at Ser473 but does not affect the level of total Akt1. The relationship between lysate protein concentration from untreated and IGF-1-treated MCF7 cells and the absorbance at 450 nm using the FastScan™ Phospho-Akt1 (Ser473) ELISA Kit #54336 is shown in the upper figure. The corresponding western blots using Akt1 antibody (left panel) and phospho-Akt (Ser473) antibody (right panel) are shown in the lower figure. After serum starvation, MCF7 cells were treated with 100 ng/ml IGF-1 #8917 for 5 minutes at 37°C and then lysed.

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Product Includes Volume (with Count) Solution Color
Akt1 Rabbit Capture mAb 1 x 1 ea Green (Lyophilized)
Phospho-Akt (Ser473) Rabbit HRP-linked mAb 1 x 1 ea Red (Lyophilized)
FastScan™ ELISA Capture Antibody Diluent 1 x 3 ml Green
FastScan™ ELISA HRP Antibody Diluent 1 x 3 ml
TMB Substrate 7004 1 x 11 ml
STOP Solution 7002 1 x 11 ml
Sealing Tape 1 x 1 ea
ELISA Wash Buffer (20X) 1 x 25 ml
FastScan™ ELISA Cell Extraction Buffer (5X) 1 x 10 ml
FastScan™ ELISA Cell Extraction Enhancer Solution (50X) 1 x 1 ml
FastScan™ ELISA Kit #54336 Positive Control 2 x 1 ea

FastScan™ ELISA Protocol

A. Solutions and Reagents

NOTE: Prepare solutions with deionized/purified water or equivalent.
Prepare only as much reagent as needed on the day of the experiment.

  1. FastScan™ ELISA Microwell Strip Plate, 96 well (#53257): Bring all to room temperature before opening bag/use. Unused microwell strips should be returned to the original re-sealable bag containing the desiccant pack and stored at 4°C.
  2. 1X ELISA Wash Buffer: Prepare by diluting ELISA Wash Buffer (20X) (included in each kit) to 1X with deionized water.
  3. 1X Cell Extraction Buffer: Prepare by diluting FastScan™ ELISA Cell Extraction Buffer (5X) #69905 and FastScan™ ELISA Cell Extraction Enhancer Solution (50X) #25243* to 1X with deionized water. This buffer can be stored at 4°C for short-term use (1-2 weeks). To make 10 mL 1X Cell Extraction Buffer, combine 7.8 mL deionized water, 2 mL FastScan™ ELISA Cell Extraction Buffer (5X), and 200 μL FastScan™ ELISA Cell Extraction Enhancer Solution (50X). Alternatively, Enhancer Solution may be added to the Cell Extraction Buffer after extraction of cells or tissue. When using the 1X Cell Extraction Buffer as a sample diluent for the assay, it is recommended to equilibrate it to room temperature prior to use.
  4. *IMPORTANT: The provided FastScan™ ELISA Cell Extraction Enhancer Solution (50X) may precipitate when stored at 4°C. To dissolve, warm briefly at 37°C and mix gently. The FastScan™ ELISA Cell Extraction Enhancer Solution (50X) can be stored at room temperature to avoid precipitation.

    NOTE: The 1X Cell Extraction Buffer contains phosphatase inhibitors. Protease inhibitors should be added to the 1X Cell Extraction Buffer immediately prior to lysing cells. Additional phosphatase inhibitors can also be added (e.g. Protease/Phosphatase Inhibitor Cocktail (100X) #5872, not supplied).

  5. FastScan™ ELISA Capture Antibody Diluent: Green diluent for reconstitution of the Capture Antibody.
  6. FastScan™ ELISA HRP Antibody Diluent: Diluent (amber bottle) for reconstitution of the HRP-linked Antibody. Protect from light.
  7. 4X Capture Antibody: Reconstitute lyophilized Capture Antibody (green colored cake) with 3 mL FastScan™ ELISA Capture Antibody Diluent (green diluent). Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. For best results, use immediately following antibody reconstitution. Unused reconstituted 4X Capture Antibody may be stored for up to 4 weeks at 4°C, although there may be some loss of signal compared to freshly reconstituted antibody.
  8. 4X HRP-linked Antibody: Reconstitute lyophilized HRP-linked Antibody (red colored cake) with 3 mL FastScan™ ELISA HRP Antibody Diluent. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. For best results, use immediately following antibody reconstitution. Unused reconstituted 4X HRP-linked Antibody may be stored for up to 4 weeks at 4°C protected from light, although there may be some loss of signal compared to freshly reconstituted antibody.
  9. Antibody Cocktail: Combine equal volumes of the reconstituted 4X Capture and 4X HRP-linked Antibodies immediately prior to assay and mix. To make 6 mL of the Antibody Cocktail (enough for 1x 96-well plate), combine 3 mL 4X Capture Antibody with 3 mL 4X HRP-linked Antibody.
  10. Positive Control: Reconstitute 1 vial of lyophilized Positive Control by adding 250 μL deionized water. Mix thoroughly and gently, hold at room temperature for 1 minute and then follow the steps outlined below in the "Test Procedure" section. Positive Controls are recommended to be used immediately after reconstituting in deionized water, however remaining material may be stored at -80°C (there may be some loss of the positive control signal if freeze/thawed). Positive Controls are supplied as a control reagent, not as an absolute quantitation measure.
  11. TMB Substrate (#7004): Bring to room temperature before use.
  12. STOP Solution (#7002): Bring to room temperature before use.

B. Preparing Cell Lysates

For adherent cells

  1. Aspirate media when the culture reaches 80-90% confluence.
  2. Remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 mL ice-cold 1X Cell Extraction Buffer (recommended to supplement with protease inhibitors and additional phosphatase inhibitors as needed) 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 5 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 (~1200 rpm) when the culture reaches 0.5-1.0 x 106 viable cells/ml.
  2. Wash once with ice-cold 1X PBS.
  3. Cells harvested from 50 mL of growth media can be lysed in 2.0 mL of 1X Cell Extraction Buffer (recommended to supplement with protease inhibitors and additional phosphatase inhibitors as needed).
  4. Sonicate lysates on ice.
  5. Microcentrifuge for 5 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. Test Procedure

NOTE: Equilibrate all materials and prepared reagents to room temperature prior to running the assay.

  1. Prepare all reagents as indicated above (Section A).
  2. Samples should be undiluted or diluted with 1X Cell Extraction Buffer to a 2X protein concentration in order to achieve a final 1X protein concentration upon addition of the antibody cocktail. Individual datasheets for each kit provide a sensitivity curve that serves as a reference for selection of an appropriate starting lysate concentration. The sensitivity curve shows typical results across a range of lysate concentration points.
  3. Add 50 μL of each sample or Positive Control to the appropriate wells.
  4. Add 50 μL of the Antibody Cocktail to each well.
  5. Seal the plate with the supplied sealing tape and incubate for 1 hour at room temperature on a plate shaker set to 400 rpm (moderate agitation).
  6. Gently remove the tape and wash wells:
    1. Discard plate contents into a receptacle.
    2. Wash 3 times with 1X ELISA Wash Buffer, 200 μL each time for every well. After each wash, aspirate or decant from wells. Invert the plate and blot it against clean paper towels 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.
  7. Add 100 μL of TMB Substrate to each well. Seal with tape and incubate the plate in the dark for 15 min at room temperature on a plate shaker (400 rpm, moderate agitation) or alternatively for 10 min at 37°C without shaking.
  8. Add 100 μL of STOP Solution to each well. Shake gently for a few seconds.
  9. NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.

  10. Read results:
    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 May 2018

The FastScan™ Phospho-Akt1 (Ser473) ELISA Kit is a sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Akt1 when phosphorylated at Ser473. To perform the assay, sample is incubated with a capture antibody conjugated with a proprietary tag and a second detection antibody linked to HRP, forming a sandwich with phospho-Akt1 (Ser473) in solution. This entire complex is immobilized to the plate via an anti-tag antibody. The wells are then washed to remove unbound material. TMB is then added. The magnitude of observed signal is proportional to the quantity of phospho-Akt1 (Ser473). Antibodies in kit are custom formulations specific to kit.

The FastScan™ Phospho-Akt1 (Ser473) ELISA Kit detects endogenous levels of Akt1 when phosphorylated at Ser473 as shown in Figure 1. This kit 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, Rat, Monkey

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. Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.
  5. Sarbassov, D.D. et al. (2005) Science 307, 1098-101.
  6. Jacinto, E. et al. (2006) Cell 127, 125-37.
  7. Cardone, M.H. et al. (1998) Science 282, 1318-21.
  8. Brunet, A. et al. (1999) Cell 96, 857-68.
  9. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
  10. Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.
  11. Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.
  12. Hajduch, E. et al. (2001) FEBS Lett 492, 199-203.
  13. Cross, D.A. et al. Nature 378, 785-9.
  14. Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511.
  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
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
FastScan™ ELISA is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
U.S. Patents 9,086,407, 9,261,500, and 9,476,874, foreign equivalents, and child patents deriving therefrom.

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54336C
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