Jhdavis::Immunoprecip

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(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...)
Current revision (16:30, 30 April 2009) (view source)
 
Line 2: Line 2:
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

Current revision

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

Personal tools