Immunoprecipitation Protocol

IMMUNOPRECIPITATION PROTOCOL

 Preparation of worm lysate:

 

Add 2 ml of PLC buffer to 0.25 ml of worm pellet.

Break up the worms by sonicating.

Spin 10 min @ 13000 g in microfuge, save supernatant.

 

Quantitate protein in lysate using Pierce BCA kit.

 

Wash 100 ml (actually 10 ml of beads since it comes as a 10% suspension) of Protein A Sepharose in PLC buffer. Spin 3 min @ 13000 g in microfuge.

 

Immunoprecipitate using 1 mg of lysate protein (may have to use more depending on the protein you’re looking at) mixed with 5 mg of antibody and 10 ml of the washed Protein A Sepharose. Total volume of the mixture should be 1 ml.

Incubate on a rocker platform at 4ºC for 1 hr.

 

Wash 3x with 1 ml of PLC buffer.

Add 50 ml 1x sample buffer.

Boil 5 min.

Load 20 ml on  an SDS PAGE gel.

 

 

 

 

PLC buffer:

 

50 mM Hepes                          Also added to this the following inhibitors:

150 mM NaCl

10% glycerol                            1 mM sodium orthovanadate

1% Triton X-100                      1 mM PMSF

1.5 mM MgCl2                 10 mg/ml leupeptin

1 mM EGTA                            2 mM benzamidine

10 mM NaPPi

10 mM NaF