|H All||Endogenous||Rabbit IgG|
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.
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
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*.
Proceed to one of the following specific set of steps.
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).
posted December 2007
Protocol Id: 27
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
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.)
Human, All Species Expected
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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, 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 CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at email@example.com.
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|5501S||400 µl||$ 297.0|