Immunoprecipitation sample buffer
Lysate Preparation The quality of the sample used for immunoprecipitation critically depends on the right lysis buffer. The ideal lysis buffer will stabilize native protein conformation, inhibit enzymatic activity, prevent antibody binding site denaturation and ensure maximum release of proteins from the cells or tissue for capture and analysis.I. Sample Preparation and Immunoprecipitation A. Materials Required 1X PBS Magnetic Myc Immunoprecipitation Kit (Cat. No. ) NaOH, 1 l at a time, until the sample buffer turns blue. Alternative elution (1X SDS): Add 20 l of 1X SDS sample buffer to the beads from step 7. immunoprecipitation sample buffer
Immunoprecipitation Protocol (For Analysis By Western Immunoblotting): Resuspend the pellet with 20 l 3X SDS sample buffer. Vortex, then microcentrifuge for 30 seconds. Heat the sample to C for 25 minutes and microcentrifuge for 1 minute at 14, 000 X g.
IMMUNOPRECIPITATION (IP) PROTOCOL Immunoprecipitation is a method that enables the purification of a protein. An antibody for the protein of interest is incubated with a cell extract so that the antibody will bind the protein in solution. The antibodyantigen complex will then be pulled out of the sample using protein AGcoupled agarose beads. Immunoprecipitation is a method that enables the purification of a protein. An antibody for the protein of interest is incubated with a cell extract enabling the antibody to bind to the protein in solution. The antibodyantigen complex is then pulled out of the sample using protein AGcoupled agarose beads. This isolates the protein ofimmunoprecipitation sample buffer proteins and is sufficient for sample preparation and immunoprecipitation of up to 50 reactions. II. Sample Preparation and Immunoprecipitation A. Required Materials Supplied in Kit Magnetic Beads Immunoprecipitation Buffer Set (Cat. No. ) o Lysis Buffer (110 ml) o Wash Buffer (2 x 100 ml) o IP Elution Buffer (2 ml)