Jhdavis::Immunoprecip: Difference between revisions
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(New page: 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 freez...) |
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20 mL culture, mid-log phase | 20 mL culture, mid-log phase | ||
centrifuge 5 min, 5000 rmp | centrifuge 5 min, 5000 rmp | ||
resuspend in 2 mL of 10 mM tris pH 8.0, 1 mM EDTA, 1mg/mL lysozyme | resuspend in 2 mL of 10 mM tris pH 8.0, 1 mM EDTA, 1mg/mL lysozyme | ||
freeze/thaw | freeze/thaw | ||
sonication | sonication | ||
centrifuge 10 min, 14,000 rmp | centrifuge 10 min, 14,000 rmp | ||
dilute 50 uL sup with 950 uL PBS | dilute 50 uL sup with 950 uL PBS | ||
add antibody and incubate 30 min, 4 C | add antibody and incubate 30 min, 4 C | ||
add proteinA-sepharose (35 uL of 1:1 beads:PBS suspension) | add proteinA-sepharose (35 uL of 1:1 beads:PBS suspension) | ||
1.5 h at 4 C | 1.5 h at 4 C | ||
centrifuge 1 min, 1000 rpm | centrifuge 1 min, 1000 rpm | ||
wash 3 times with PBS, 1% tritonx100, 5 mM EDTA | wash 3 times with PBS, 1% tritonx100, 5 mM EDTA | ||
Latest revision as of 13:30, 30 April 2009
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
