Jhdavis::Immunoprecip

Immunoprecipitation (sherman et al. 1992 EMBO J 11: 71-77)

20 mL culture, mid-log phase

centrifuge 5 min, 5000 rmp

resuspend in 2 mL of 10 mM tris pH 8.0, 1 mM EDTA, 1mg/mL lysozyme

freeze/thaw

sonication

centrifuge 10 min, 14,000 rmp

dilute 50 uL sup with 950 uL PBS

add antibody and incubate 30 min, 4 C

add proteinA-sepharose (35 uL of 1:1 beads:PBS suspension)

1.5 h at 4 C

centrifuge 1 min, 1000 rpm

wash 3 times with PBS, 1% tritonx100, 5 mM EDTA