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PhosphoSitePlus® Resource

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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M Endogenous 60 Rabbit IgG
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Immunohistochemistry (Frozen)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing cytoplasmic and nuclear localization, using Phospho-Akt (Ser473) (736E11) Rabbit mAb.

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Immunohistochemistry (Frozen)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-Akt (Ser473) (736E11) Rabbit mAb.

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Immunohistochemistry (Frozen)

Immunohistochemical analysis using Phospho-Akt (Ser473) (736E11) Rabbit mAb on SignalSlide (R) Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right).

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Immunohistochemistry (Frozen)

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma, using Phospho-Akt (Ser473) (736E11) Rabbit mAb preincubated with an irrelevant peptide (left) or Phospho-Akt (Ser473) Blocking Peptide (#1140) (right).

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Immunohistochemistry (Frozen)

Immunohistochemical analysis of paraffin-embedded MDA-MB-468 xenograft, using Phospho-Akt (Ser473) (736E11) Rabbit mAb (left) or PTEN (138G6) Rabbit mAb #9559 (right). MDA-MB-468 cells lack PTEN. Note the presence of P-Akt in the PTEN deficient cells.

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Immunohistochemistry (Frozen)

Immunohistochemical analysis of frozen U-87MG xenograft, showing predominantly cytoplasmic localization using Phospho-Akt (Ser473)(736E11) Rabbit mAb.

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Immunohistochemistry (Paraffin)

A. Solutions and Reagents

  1. Xylene
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%)
  3. Deionized water (dH2O)
  4. Hematoxylin (optional)
  5. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
  6. SignalStain® Antibody Diluent (#8112).
  7. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH2O.
  8. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  10. Detection System: VECTASTAIN® Elite ABC, including biotinylated secondary antibody (Vector Laboratories).
  11. Substrate: Vector® NovaRED™ (Vector Laboratories).
  12. Hematoxylin: Hematoxylin (#14166).
  13. Mounting Medium: SignalStain® Mounting Medium (#14177).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 minutes each.
    2. Incubate sections in two washes of 100% ethanol for 10 minutes each.
    3. Incubate sections in two washes of 95% ethanol for 10 minutes each.
  2. Wash sections twice in dH2O for 5 minutes each.

C. Antigen Unmasking

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

D. Staining

  1. Wash sections in dH2O three times for 5 minutes each.
  2. Incubate sections in 3% hydrogen peroxide for 10 minutes.
  3. Wash sections in dH2O twice for 5 minutes each.
  4. Wash sections in wash buffer for 5 minutes.
  5. Block each section with 100-400 µl of preferred blocking solution for 1 hour at room temperature.
  6. Remove blocking solution and add 100-400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
  7. Prepare ABC solution per manufacturer's recommendations.
  8. Remove primary antibody and wash section three times with wash buffer for 5 minutes each.
  9. Add 100-400 µl biotinylated secondary antibody, diluted in TBST per manufacturer’s recommendation, to each section. Incubate 30 minutes at room temperature.
  10. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
  11. Cover sections with 100-400 µl pre-mixed ABC solution as needed and incubate in a humidified chamber for 30 min at room temperature.
  12. Wash section three times with wash buffer for 5 min each.
  13. Prepare Vector® NovaRED™ per manufacturer's recommendations.
  14. Apply 100-400 µl substrate to each section and monitor closely. 1-10 minutes generally provides an acceptable staining intensity.
  15. If desired, counterstain sections with hematoxylin (#14166).
  16. Wash sections in dH2O two times for 5 minutes each.
  17. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 seconds each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
    3. Repeat in xylene, incubating sections two times for 10 seconds each.
  18. Mount sections with coverslips and mounting medium (#14177).

posted June 2005

revised March 2016

Protocol Id: 303

Immunohistochemistry (Frozen)

A. Solutions and Reagents

  1. Xylene
  2. Ethanol (anhydrous denatured, histological grade 100% and 95%)
  3. Deionized water (dH2O).
  4. Fixative: 3% formaldehyde.
    1. To prepare 100 ml, add 18.75 ml 16% formaldehyde to 81.25 ml 1X PBS.
  5. Wash Buffer: 1X Tris Buffered Saline (TBS).

    To prepare 1L 1X TBS add 100 ml 10X Tris Buffered Saline (#12498) to 900 ml dH2O, mix.

  6. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  7. Blocking Solution: 1X TBS/0.3% Triton X-100/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. 1X TBS/0.3% Triton X-100/5% Normal Goat Serum: add 500 µl Normal Goat Serum (#5425) and 30 µl Triton X-100 to 9.5 ml 1X TBS.
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O, add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  8. Antibody Diluent 1X TBS/0.3% Triton™ X-100/5% Normal Goat Serum: add 500 µl Normal Goat Serum (#5425) and 30 µl Triton™ X-100 to 9.5 ml 1X TBS.
  9. Detection System: VECTASTAIN® Elite ABC, including biotinylated secondary antibody (Vector Laboratories).
  10. Substrate: Vector® NovaRED™ (Vector Laboratories).
  11. Hematoxylin: Hematoxylin (#14166).
  12. Mounting Medium: SignalStain® Mounting Medium (#14177).

B. Sectioning

  1. For tissue stored at -80°C: remove from freezer and equilibrate at -20°C for approximately 15 minutes before attempting to section. This may prevent cracking of the block when sectioning.
  2. Section tissue at a range of 6-8 µm and place on positively charged slides.
  3. Allow sections to air dry on bench for a few minutes before fixing (this helps sections adhere to slides).

C. Fixation

  1. Fix sections in 3% formaldehyde for 15 min at room temperature. Proceed with staining procedure immediately (Section D).

D. Staining

  1. Wash sections in wash buffer twice for 5 minutes.
  2. Incubate for 10 minutes at room temperature in 3% Hydrogen Peroxide.
  3. Wash sections in wash buffer twice for 5 minutes.
  4. Block each section with 100-400 µl of preferred blocking solution for one hour at room temperature.
  5. Remove blocking solution and add 100-400 µl primary antibody diluted in recommended antibody diluent to each section.
  6. Incubate overnight at 4°C
  7. Prepare ABC solution per manufacturer's recommendations.
  8. Remove primary antibody and wash section three times with wash buffer for 5 minutes each.
  9. Add 100-400 µl biotinylated secondary antibody, diluted in wash buffer per manufacturer's recommendation, to each section. Incubate 30 minutes at room temperature.
  10. Remove secondary antibody solution and wash sections three times with wash buffer for 5 min each.
  11. Cover sections with 100-400 µl pre-mixed ABC solution as needed and incubate in a humidified chamber for 30 min at room temperature.
  12. Wash section three times with wash buffer for 5 min each.
  13. Prepare Vector® NovaRED™ per manufacturer's recommendations.
  14. Apply 100-400 µl substrate to each section and monitor closely. 1-10 min generally provides an acceptable staining intensity.
  15. Immerse slides in dH2O.
  16. If desired, counterstain sections in hematoxylin (#14166).
  17. Wash sections in dH2O two times for 5 min each.
  18. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 seconds each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
    3. Repeat in xylene, incubating sections two times for 10 seconds each.
  19. Mount sections with coverslips and mounting medium (#14177).

posted January 2006

revised March 2016

Protocol Id: 330

Product Usage Information

Application Dilutions
Immunohistochemistry (Paraffin) 1:50
Immunohistochemistry (Frozen) 1:50

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

Phospho-Akt (Ser473) (736E11) Rabbit mAb detects Akt1 only when phosphorylated at serine 473, and Akt2 and Akt3 only when phosphorylated at equivalent sites.


Species Reactivity: Human, Mouse
Species predicted to react based on 100% sequence homology: Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser473 of mouse Akt.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


Entrez-Gene Id 207 , 208 , 10000
Swiss-Prot Acc. P31749 , P31751 , Q9Y243


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 5,675,063.

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Phospho-Akt (Ser473) (736E11) Rabbit mAb