Western blot analysis of extracts from COS cells transfected with Myc-Chorionic Somatomammotropin Hormone-Like 1 (CSL) protein (lane 1). Immunoprecipitation of transfected COS lysates with Mouse IgG (Sepharose® Bead Conjugate) (lane 2) or Myc-Tag (9B11) (Sepharose® Bead Conjugate) Mouse mAb #3400 (lane 3). Blot was probed with Myc Tag (71D10) Rabbit mAb #2278.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.
Normal mouse IgG is not expected to recognize any specific antigen.
Normal mouse IgG was isolated by affinity chromatography from naive mouse serum.
This Cell Signaling Technology normal mouse IgG (whole molecule) containing predominantly IgG1 and IgG2a isotypes and small amounts of IgG2b and IgG3 is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads. Mouse IgG (Sepharose® Bead Conjugate) is useful to determine non-specific immunoprecipitation complexes.
Control antibodies are used to estimate the non-specific binding of target primary antibodies due to Fc receptor binding or other protein-protein interactions.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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|3420S||400 µl (40 immunoprecipitations)||$109.00.0|