Immunoprecipitation (IP) of COX IV from HeLa cells using COX IV (4D11-B3-E8) Mouse mAb #11967 and Protein G Agarose Beads. Western blot analysis was performed on the IP pellet (lane 1) and supernatant (lane 2) using COX IV (3E11) Rabbit mAb #4850.
Immunoprecipitation (IP) of Akt from 3T3 cells using Akt (pan) (40D4) Mouse mAb #2920 and Protein G Agarose Beads. Western blot analysis was performed on the IP pellet (lane 1) and supernatant (lane 2) using Akt (pan) (C67E7) Rabbit mAb #4691.
Vortex tube briefly to resuspend the beads. Add 20-40 μl of bead slurry to each immunoprecipitation (IP) reaction. For bead washing and subsequent elution of immunocomplexes, the beads can be separated from solution by a brief 1 minute centrifugation in a microcentrifuge at 6,000 rpm. Resuspend the beads in solution by gently vortexing or rocking the tube. Please follow CST's recommended IP protocol to perform IP followed by western blot.
Supplied as a 50% slurry in 20% ethanol solution. Store at 4°C. This product is stable for 12 months.
This protocol is intended for immunoprecipitation of native proteins utilizing Protein G agarose beads for subsequent analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to 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 October 2016
revised April 2018
Protocol Id: 1244
Protein G Agarose Beads are an affinity matrix for the small-scale isolation of immunocomplexes from immunoprecipitations (IP assays). Protein G is covalently coupled to agarose beads. Protein G exhibits high affinity for subclasses of IgG from many species (including human, rabbit, mouse, rat, and sheep) and can be used for immunoprecipitation assays with these antibodies.
Bead Diameter: ~50-150 micron per bead
Binding Capacity: ~20 mg human IgG/ml
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