Immunoprecipitation of Jurkat cell extracts using Akt (pan) (C67E7) Rabbit mAb #4691. Extracts were incubated with either Anti-Rabbit IgG F(ab')2 Fragment (Sepharose® Bead Conjugate) (lane 1) or Protein A Beads (lane 2). The western blot was probed using Akt (pan) (40D4) Mouse mAb #2920.
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads. Anti-rabbit IgG F(ab') 2 Fragment (Sepharose® Bead Conjugate) is useful for the immunoprecipitation of antibodies raised in rabbit.
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, Rabbit IgG and Rabbit serum. No reaction was observed against anti-pepsin, anti-goat IgG Fc, or human or mouse serum proteins.
Anti-rabbit IgG F(ab')2 Fragment is produced by immunizing goats with rabbit IgG whole molecules. The Anti-rabbit IgG F(ab')2 Fragment is prepared from monospecific antiserum by immunoaffinity chromatography using Rabbit IgG coupled to agarose beads followed by solid phase adsorption to remove any unwanted reactivities, pepsin digestion, and chromatographic separation.
Anti-rabbit IgG F(ab')2 Fragment (Sepharose Bead Conjugate) is ideal for immunoprecipitation. This secondary antibody will detect rabbit 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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