Immunoprecipitation buffers and protocols免疫沉淀实验缓冲液及方法

Steven Finkbeiner, Departments of Neurology and Physiology, UCSF
http://gweb1.ucsf.edu/labs/finkbeiner/Protocol/protocol_list/Imm_precip.htm
 
Prepare pG agarose solution by washing with lysis buffer* and spinning down at 3000 rpm for 2 min (repeat once)

Prepare largest samples possible (all samples should have equal ug) and add lysis

buffer so that all samples are equal in volume

Preincubate samples in pG solution for 1 hour on 4 C agitator

Spin down for 1 min at 13000 rpm

Transfer supernatant to new tube

Repeat preceding 2 steps

Ad Ab and incubate for 1 hour on 4 C agitator

Add pG solution

Incubate for 1 hour on 4 C agitator

Spin for 3 min 3000 rpm at 4 C

Remove supernatant

Wash with 500 ul lysis buffer

Repeat preceding 3 steps (spin, remove supernatant, wash)

Resuspend bead with 30ul SBS 1 + DTT 100 mM

for 500 ul:

100 ul SBS 5X

50 ul DTT 1M

350 dH20

*Lysis Buffer:

10 ml TGEK100

PMSF (1/200)

Benzamidine (1/200)

Leupeptine (1/500)

Aprotinin (1/200)

NP40 1%