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Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) Rabbit mAb (Sepharose® Bead Conjugate) #5501
Immunoprecipitation of Jurkat cell extracts, untreated (-) or treated with Calyculin A #9902 (+), using Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) Rabbit mAb (Sepharose Bead Conjugate) and Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose Bead Conjugate) #3423. The western blot was probed using Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) Rabbit mAb #2325. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.Learn more about how we get our images
Gallery: Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) Rabbit mAb (Sepharose® Bead Conjugate) #5501
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
- 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.
- Microcentrifuge for 30 seconds at 4°C. Wash pellet five times with 500 μl of 1X cell lysis buffer. Keep on ice during 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 twice with 500 µl 1X kinase buffer. Keep on ice.
- 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: 27
Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) Rabbit mAb (Sepharose Bead Conjugate) detects phospho-serine in a PXS*P or S*PXR/K motif, as well as a PXS*PXR/K motif. The antibody is phospho-specific, and does not react with phospho-threonine- or phospho-tyrosine-containing peptides/proteins. (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.)
Monoclonal antibody is produced by immunizing animals with synthetic phospho-MAPK/CDK substrate peptides.
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated sepharose beads. Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) Rabbit mAb (Sepharose Bead Conjugate) is useful for the immunoprecipitation of Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K). The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) Rabbit mAb #2325.
The MAPK and CDK families of serine/threonine protein kinases play important roles in proliferation and cell cycle control. These kinases phosphorylate threonine or serine followed by a proline residue (1-3). MAPK phosphorylates substrates with the consensus sequence PX(S/T)P, and CDKs phosphorylate substrates containing the consensus sequence (S/T)PXR/K. Cell Signaling Technology has developed antibodies that bind to phospho-threonine followed by proline, motifs PXS*/T*P and/or S*PXR/K, for use in the study and discovery of new MAPK and CDK substrates (4,5).
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.