SDS-PAGE gels and western blotting

9/23/04

1. Harvesting samples

How you harvest samples for western depends a great deal on the abundance and localization of the protein, its sensitivity to proteolysis or de/phosphorylation, and your projected application. Included here are some recipes and techniques for harvesting from which to choose.

RIPA buffer:

1% Tx100 10mL 10%

0.5% DOC 5mL 10%

0.1% SDS 0.5mL 20%

150mM NaCl 3mL 5M

50mM Tris pH 7.8 2.5mL 2M

1mM EDTA 0.2mL 0.5M

78.8mL ddH2O

Add NaF 1:1000, Aprotinin 1:100, PMSF 1:100 just before use