Jhdavis::Immunoprecip
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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