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10001
Phospho-Akt Substrate (RXRXXS*/T*) (23C8D2) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

Phospho-Akt Substrate (RXRXXS*/T*) (23C8D2) Rabbit mAb #10001

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  1. WB
Western Blotting Image 1: Phospho-Akt Substrate (RXRXXS*/T*) (23C8D2) Rabbit mAb

Western blot analysis of extracts from MKN-45 cells, untreated or treated with c-Met kinase inhibitor SU11274 (1 µM, 2 hr), using Phospho-Akt Substrate (RXRXXS*/T*) (23C8D2) Rabbit mAb. Western blot image was obtained using the Odyssey® Infrared Imaging System (LI-COR® Biotechnology).

Western Blotting Image 2: Phospho-Akt Substrate (RXRXXS*/T*) (23C8D2) Rabbit mAb

Western blot analysis of extracts from A-431 cells, untreated or treated with Human Epidermal Growth Factor (hEGF) #8916 (10 ng/ml, 20 min), using Phospho-Akt Substrate (RXRXXS*/T*) (23C8D2) Rabbit mAb (upper), or Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (lower). Western blot images were obtained using the Odyssey® Infrared Imaging System (LI-COR® Biotechnology).

To Purchase # 10001S
製品番号 サイズ 価格 在庫
10001S
100 µl

Supporting Data

REACTIVITY All
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Rabbit 

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Usage Information

Application Dilution
Western Blotting 1:1000
Peptide ELISA (DELFIA) 1:1000

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.

Protocol

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Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.

A. Solutions and Reagents

From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit

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. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised June 2020

Protocol Id: 10

ELISA Peptide

A. Solutions and Reagents

  1. Carbonate Buffer: 15 mM Na2CO3, 35 mM NaHCO3, 0.2 g/L NaN3 (pH 9.6). Use 1 μM synthetic peptide in carbonate buffer.
  2. 10X Phosphate Buffered Saline (PBS): To prepare 1 L add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phosphate, dibasic (Na2HPO4) and 2.4 g potassium phosphate, monobasic (KH2PO4) to 1 L dH2O. Adjust pH to 7.4.
  3. Wash Buffer: 1X PBS containing 0.05% Tween-20 (PBST)
  4. Blocking Buffer: 10 mg/ml bovine serum albumin (BSA) in PBST
  5. Antibody Dilution Buffer: 3% BSA in PBST
  6. DELFIA® Europium-labeled Anti-mouse IgG for mouse primary antibodies or Anti-rabbit IgG (PerkinElmer Life Sciences #AD0124) for rabbit primary antibodies.
  7. DELFIA® Enhancement Solution (PerkinElmer Life Sciences #1244-105)

(DELFIA® is a registered trademark of PerkinElmer, Inc.)

B. Protocol

  1. Coat the wells of a 96-well microtiter plate with 100 μl of 1 μM synthetic peptide in carbonate buffer by incubating overnight at 4°C or for 2 to 6 hrs at 37°C. If the peptide does not bind or absorb, try other buffers in the pH 4–8 range.
  2. Wash plate three times 200 μl/well with wash buffer.
  3. Block plate with 200 μl/well blocking buffer for 1 hr at 37°C. Wash plate three times with wash buffer. (May leave dry plate at 4°C for 1–2 months if desired.)
  4. Prepare appropriate dilution of primary antibody with antibody dilution buffer. Add 100 μl to wells and incubate at 37°C for 1 hr.
  5. Wash three times with wash buffer.
  6. Add 67 ng/well DELFIA Europium-labeled Anti-mouse IgG or Anti-rabbit IgG, diluted in 100 μl/well antibody dilution buffer. Incubate at room temperature for 30 min, on gentle shaker.
  7. Wash three times with wash buffer.
  8. Add 100 μl enhancement solution and incubate at room temperature for 5 min. Read plate at 615 nm with an appropriate time-resolved plate reader.

posted June 2005

revised September 2007

Protocol Id: 34

Specificity / Sensitivity

Phospho-Akt Substrate (RXRXXS*/T*) (23C8D2) Rabbit mAb recognizes endogenous proteins containing phospho-Ser/Thr preceded by Arg at positions -5 and -3 in a manner largely independent of the surrounding amino acid sequence. Minor cross-reactivity is observed for proteins that contain phospho-Ser/Thr preceded by Arg at position -3 only. No cross-reactivity is observed with the corresponding nonphosphorylated sequences or with other phospho-Ser/Thr-containing motifs.

Species Reactivity:

All Species Expected

Source / Purification

Monoclonal antibody is produced by immunizing animals with an Akt substrate peptide library.

Background

An important class of kinases, referred to as Arg-directed kinases or AGC-family kinases, includes cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), protein kinase C, Akt, and RSK. These kinases share a substrate specificity characterized by Arg at position -3 relative to the phosphorylated Ser or Thr (1,2). Akt plays a central role in mediating critical cellular responses including cell growth and survival, angiogenesis, and transcriptional regulation (3-5). While a number of Akt substrates are known (such as GSK-3, Bad, and caspase-9) many important substrates await discovery. Akt phosphorylates substrates only at Ser/Thr in a conserved motif characterized by Arg at positions -5 and -3 (6). Phospho-Akt substrate-specific antibodies from Cell Signaling Technology are powerful tools for investigating the regulation of phosphorylation by Akt and other Arg-directed kinases, as well as for high throughput kinase drug discovery.

  1. Montminy, M. (1997) Annu Rev Biochem 66, 807-22.
  2. Pearson, R.B. and Kemp, B.E. (1991) Methods Enzymol 200, 62-81.
  3. Marte, B.M. and Downward, J. (1997) Trends Biochem Sci 22, 355-8.
  4. Jiang, B.H. et al. (2000) Proc Natl Acad Sci USA 97, 1749-53.
  5. Scheid, M.P. and Woodgett, J.R. (2000) Curr Biol 10, R191-4.
  6. Alessi, D.R. et al. (1996) FEBS Lett 399, 333-8.

使用に関する制限

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For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
LI-COR is a registered trademark of LI-COR, Inc.
Odyssey is a registered trademark of LI-COR, Inc.
Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at [email protected] For information regarding commercial licensing terms please contact CST Pharma Services Department at [email protected]
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