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Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (Magnetic Bead Conjugate) #8050
Immunoprecipitation of Jurkat cell lysates, untreated or treated with Calyculin A, using Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (Magnetic Bead Conjugate) (lane 1 and 2) and Rabbit (DA1E) mAb IgG XP® Isotype Control (Magnetic Bead Conjugate) #8726 (lane 3 and 4). The western blot was probed using Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (HRP Conjugate) #6950.Learn more about how we get our images
Gallery: Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (Magnetic Bead Conjugate) #8050
Immunoprecipitation for Analysis by Western Blotting
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
- 20X Phosphate Buffered Saline (PBS): (#9808).
10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.
- 3X SDS Sample Buffer: (#7722) 187.5 mM Tris-HCl (pH 6.8 at 25°C), 6% w/v SDS, 30% glycerol, 150 mM DTT, 0.03% w/v bromophenol blue
- 6-Tube Magnetic Separation Rack: (#7017).
- 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH2O, mix.
- ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.
B. Preparing Cell Lysates
- Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
- To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS.
- Remove PBS and add 0.5 ml 1X ice-cold cell lysis buffer to each plate (10 cm) and incubate the plates on ice for 5 minutes.
- Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.
- Sonicate samples on ice three times for 5 seconds each.
- Microcentrifuge for 10 minutes at 4°C, 14,000 x g, and transfer the supernatant to a new tube. If necessary, lysate can be stored at –80°C.
- Take 200 μl cell lysate and add 10 µl of the immobilized antibody, incubate with rotation overnight at 4°C.
- Wash pellet by using magnetic rack to pellet beads, then discard supernatant once completely clear and add 500 µl of 1X cell lysis buffer, vortex to resuspend and wash the beads, repeat 4 more times for a total of 5 washes.
- Proceed to sample analysis by western blotting or kinase activity (section D).
D. Sample Analysis
Proceed to one of the following specific set of steps.
For Analysis by Western Immunoblotting
- Resuspend the pellet with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
- Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
- Load the sample (15–30 µl) on a 4–20% gel for SDS-PAGE.
- Analyze sample by western blot (see Western Immunoblotting Protocol).
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
For Analysis by Kinase Assay
- Wash pellet by using magnetic rack to pellet beads, then discard supernatant once completely clear and add 500 Âµl of 1X kinase buffer, vortex to resuspend and wash the beads, repeat 1 more time for a total of 2 washes. Keep on ice during washes.
- Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
- Incubate for 30 min at 30°C.
- Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
- Transfer supernatant containing phosphorylated substrate to another tube.
- Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g.
- Load the sample (15–30 µl) on SDS-PAGE (4–20%).
posted December 2007
protocol id: 408
Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (Magnetic Bead Conjugate) recognizes peptides and proteins containing phospho-Ser/Thr preceded by Arg at the -3 position. There is some preference observed for peptides that contain phospho-Ser/Thr preceded by Arg at both positions -5 and -3. (U.S. Patent No's.: 6,441,140; 6,982,318; 7,259,022; 7,344,714; U.S.S.N. 11,484,485; and all foreign equivalents.)Species Reactivity: Mouse, D. melanogaster
Monoclonal antibody is produced by immunizing animals with synthetic phospho-Akt substrate peptides.
This Cell Signaling Technology antibody is immobilized by the covalent reaction of formylbenzamide-modified antibody with hydrazide-activated magnetic bead.
Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (Magnetic Bead Conjugate) is useful for immunoprecipitation of phosphorylated Akt substrate proteins.
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.
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. 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 CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at firstname.lastname@example.org.