Immunoprecipitation of COS-7 cell extracts, untransfected or transfected with a construct overexpressing TrCP1-GST using GST (26H1) Mouse mAb #2624. Extracts were incubated with either Anti-Mouse IgG (H+L), F(ab')2 Fragment (Sepharose® Bead Conjugate) (lane 1 and 2) or Protein G Beads (lane 3). The western blot was probed using GST (91G1) Rabbit mAb #2625.
Immunoprecipitation of NIH/3T3 cell extracts using Akt (pan)(40D4) Mouse mAb #2920. Extracts were incubated with either Anti-Mouse IgG (H+L), F(ab')2 Fragment (Sepharose® Bead Conjugate) (lane 1) or Protein G Beads (lane 2). The western blot was probed using Akt (pan) (C67E7) Rabbit mAb #4691.
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated sepharose beads. Anti-mouse IgG (H+L), F(ab')2 Fragment (Sepharose Bead Conjugate) is useful for the immunoprecipitation of antibodies raised in mice.
Add 10-20 μl of well-vortexed beads to 200 μl of cell lysate at 1 mg/ml in 1X Cell Lysis Buffer (10X) #9803. See protocol for more details.
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.
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
Assay by immunoelectrophoresis resulted in a single precipitin arc against anti-goat serum, mouse IgG and mouse serum. No reaction was observed against anti-pepsin, anti-goat IgG Fc, or human serum proteins.
The Anti-mouse IgG (H+L), F(ab')2 Fragment is produced by immunizing goats with mouse IgG whole molecules. The Anti-mouse IgG (H+L), F(ab')2 Fragment is prepared from monospecific antiserum by immunoaffinity chromatography using mouse IgG coupled to agarose beads followed by solid phase adsorption to remove any unwanted reactivities, pepsin digestion, and chromatographic separation.
Anti-mouse IgG (H+L), F(ab')2 Fragment (Sepharose Bead Conjugate) is ideal for immunoprecipitation. This secondary antibody will detect mouse antibodies. Since this secondary antibody is made with F(ab')2 fragments, non-specific binding through Fc receptors present on cells will be eliminated.
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