Upstream / Downstream
Explore pathways related to this product.
CST Antibody Performance Guarantee
To Purchase # 8107S
|8107S||1 Kit (4 x 40 µl)||$459.00|
|$ 0. 00|
Find answers on our FAQs page.
PTM information and tools available.
SignalStain® Akt Pathway IHC Sampler Kit #8107
Immunohistochemical analysis of paraffin-embedded LNCaP cell pellets, either untreated (left) or LY294002-treated (middle) or NIH/3T3 cell pellets (right), using Phospho-Akt (Ser473) (D9E) Rabbit mAb, Akt (pan) (C67E7) Rabbit mAb, PTEN (D4.3) Rabbit mAb and Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) Rabbit mAb. Cell pellets are provided in the SignalSlide® Akt Family IHC Controls.Learn more about how we got this image
Gallery: SignalStain® Akt Pathway IHC Sampler Kit #8107
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- Ethanol, anhydrous denatured, histological grade (100% and 95%).
- Deionized water (dH2O).
- Hematoxylin (optional).
- Wash Buffer:
- 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.
- SignalStain® Antibody Diluent (#8112).
- Antigen Unmasking Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7â¢2H2O) to 1 L dH2O. Adjust pH to 6.0.
- 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
- Blocking Solution: TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
- Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
- Substrate: Vector® NovaRED™ (Vector Laboratories).
NOTE: Do not allow slides to dry at any time during this procedure.
- Deparaffinize/hydrate sections:
- Incubate sections in three washes of xylene for 5 min each.
- Incubate sections in two washes of 100% ethanol for 10 min each.
- Incubate sections in two washes of 95% ethanol for 10 min each.
- Wash sections two times in dH2O for 5 min each.
C. Antigen Unmasking
For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0; maintain at a sub-boiling temperature for 10 min. Cool slides on bench top for 30 min.
- Wash sections in dH2O three times for 5 min each.
- Incubate sections in 3% hydrogen peroxide for 10 min.
- Wash sections in dH2O two times for 5 min each.
- Wash sections in wash buffer for 5 min.
- Block each section with 100–400 µl blocking solution for 1 hr at room temperature.
- Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
- Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
- Remove antibody solution and wash sections with wash buffer three times for 5 min each.
- Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
- Wash sections three times with wash buffer for 5 min each.
- Prepare Vector® NovaRED™ per manufacturer's recommendations.
- Apply 100-400 µl substrate to each section and monitor closely. 5-15 minutes generally provides an acceptable staining intensity.
- Immerse slides in dH2O.
- If desired, counterstain sections with hematoxylin per manufacturer’s instructions.
- Wash sections in dH2O two times for 5 min each.
- Dehydrate sections:
- Incubate sections in 95% ethanol two times for 10 sec each.
- Repeat in 100% ethanol, incubating sections two times for 10 sec each.
- Repeat in xylene, incubating sections two times for 10 sec each.
- Mount sections with coverslips.
posted February 2010
revised November 2013
protocol id: 300
|Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb 4060||1 x 40 µl||Rabbit IgG|
|Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb 4858||1 x 40 µl||Rabbit IgG|
|Akt (pan) (C67E7) Rabbit mAb 4691||1 x 40 µl||Rabbit IgG|
|PTEN (D4.3) XP® Rabbit mAb 9188||1 x 40 µl||Rabbit IgG|
|SignalStain® Antibody Diluent 8112||1 x 25 ml|
|SignalSlide® Akt Family IHC Controls||x 1 Pack|
The SignalStain® Akt Pathway IHC Sampler Kit from Cell Signaling Technology allows the researcher to examine paraffin-embedded tissues or cells with antibodies directed against proteins involved in Akt signaling. Multiple approaches are used to validate each antibody for use in immunohistochemical assays. Also included in the kit are control slides that can be used to verify the performance of each antibody and a primary antibody diluent. Please see the above table for the recommended antibody diluent for each kit antibody.
Each antibody in the SignalStain® Akt Pathway IHC Sampler Kit detects endogenous levels of its target protein and does not cross-react with related proteins.Species Reactivity: Human, Mouse
Monoclonal antibody is produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding the phosphorylation site(s) of interest, and synthetic peptides corresponding to the carboxy-terminal sequence of mouse Akt or from the carboxy-terminal sequence of human PTEN.
The protein kinase Akt (also called PKB or Rac) is activated by insulin and growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (1,2). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (3) and by carboxy terminal phosphorylation at Ser473 by the mTORC2 complex composed of mammalian target of rapamycin (mTOR) in a complex with rictor and Sin1 (4,5). Akt signaling is negatively regulated via the PTEN phosphatase (6).
Akt’s several functions include inhibition of apoptosis (7-9), regulation of glycogen synthesis via GSK-3 (10), and promotion of the cell cycle (11). Akt also regulates protein synthesis by phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (mTORC1) (12). Akt also effects mTOR activity via phosphorylation and inhibition of PRAS40 (40 kDa, proline-rich protein), which binds to raptor in the mTORC1 complex and inhibits mTOR activity (13). Phosphorylation of PRAS40 by Akt at Thr246 relieves PRAS40 inhibition of mTORC1 (14), allowing protein synthesis to occur. Active of mTORC1 signals to p70 S6 kinase, which in turn phosphorylates S6 ribosomal protein. Phosphorylation of S6 ribosomal protein correlates with an increase in translation of a subset of mRNA transcripts that encode ribosomal proteins, translation elongation factors as well as regulators of cell cycle progression (15). Important S6 ribosomal protein phosphorylation sites include Ser235, Ser236, Ser240 and Ser244 within a small carboxy-terminal region (16).
Protein Specific References
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. SignalStain® is a trademark of Cell Signaling Technology, Inc. U.S. Patent No. 5,675,063.