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Immunoprecipitation of Jurkat cell extracts treated with Calyculin A #9902, using Rabbit (DA1E) mAb IgG XP® Isotype Control (Magnetic Bead Conjugate) #8726 (lane 2) or Phospho-Threonine Antibody (P-Thr-Polyclonal) (Magnetic Bead Conjugate) (lane 3). Lane 1 is 10% input. The western blot was probed using Phospho-Threonine Antibody (P-Thr-Polyclonal) (HRP Conjugate) #6949.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: 408
Supplied in PBS Buffer (pH 7.2), 0.1% Tween® 20. Store at 4°C. Do not aliquot the antibodies.
Phospho-Threonine Antibody (P-Thr-Polyclonal) (Magnetic Bead Conjugate) detects proteins and peptides phosphorylated at threonine residues in a manner largely independent of the surrounding amino acid sequence. The antibody is phospho-specific and may cross-react with some phospho-serine-containing sequences. By ELISA, it recognizes a wide variety of threonine-phosphorylated peptides, and by 2D gel Western blot analysis, it recognizes a large number of presumably threonine-phosphorylated proteins. Use of Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101) #9391 is recommended to detect proteins containing threonine followed by proline.
All Species Expected
Polyclonal antibodies are produced by immunizing animals with synthetic phospho-Thr-containing peptides. Antibodies are purified by protein A and peptide affinity chromatography.
Much of the dynamic behavior of cellular proteins, including the regulation of molecular interactions (1), subcellular localization (2), and transcriptional regulation (3) is controlled by a variety of post-translational modifications (4). Antibodies specific for these post-translational modifications are invaluable tools in the quest to understand normal and pathogenic molecular and cellular behavior.
General protein modification antibodies are designed to react with modified amino acid residues (e.g. phospho-threonine, phospho-tyrosine, acetyl-lysine, nitro-tyrosine) independently of the sequence in which they are embedded. This ability to recognize modified residues in a "context-independent" fashion gives these antibodies broad reactivities, presumably conferring upon them the ability to react with hundreds of distinct proteins. This broad pattern of reactivity makes these antibodies especially valuable in multiplex analyses and target discovery programs.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, 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.