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 μg)and add lysis

buffer so that all samples are equal in volume

Preincubate samples in pG solution for 1 hour on4℃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℃agitator

Add pG solution

Incubate for 1 hour on 4℃agitator

Spin for 3 min 3000 rpm at 4℃

Remove supernatant

Wash with 500μl lysis buffer

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

Resuspend bead with 30μl SBS1+ DTT 100 mM

for 500μl:

100μl SBS 5X

50μl DTT 1M

350 dH₂0

*Lysis Buffer:

10 ml TGEK100

PMSF (1/200)

Benzamidine (1/200)

Leupeptine (1/500)

Aprotinin (1/200)

NP40 1%