GENERAL PROTOCOL FOR IMMUNO-

PRECIPITATIONS TO BLOT

 

(NOTE:   THIS GENERAL IMMUNOPRECIPITATION PROTOCOL MAY NOT BE IDEAL FOR ALL APPLICATIONS, SEE SOMEONE IF YOU HAVE QUESTIONS ABOUT VARIATIONS)

 

1.  Step down and stimulate cells as required.  Dump media off of plates of cells into sink and place plates on tray of ice*.  Wash cells twice by gently pouring on 10-20 mls of ice-cold PBS +Calcium + Magnesium (From carboy in cold room).  Remove wash as completely as possible by shaking into sink.  Add 1 ml lysis buffer (TBS or PBS containing 1% NP-40, 10% glycerol, PMSF (1:100 from 35 mg/ml EtOH stock), 1 mg/ml each aprotinin, leupeptin, and 1-2 mM vanadate.

 

2.  Transfer plates into cold room and place on rocker.  Rock for 15-20 minutes.  Scrape cells and transfer into an eppendorf with a transfer pipette.  Spin in cold microfuge** 10 minutes, transfer supernatant into fresh tube with desired antibody.  Incubate 1 hr to overnight at 4o C.

 

 

3.  Add 50 ml of BSA-blocked protein A sepharose 6MB (1:1 slurry with PBS), put on rotator and incubate at 4o C for at least one hour.  NOTE:  you will need to cut off the end of your pipette tip so that the sepharose beads will enter your pipette tip when adding it.  Also, the beads settle quickly, so agitate the stock frequently when adding it to your samples, so that yu get equivalent amounts in each tube.

 

4.  Wash immunoprecipitates 3 times with lysis buffer at 4o C:  spin IP for 10 seconds in microfuge, aspirate supernatant, add 1 ml wash buffer.  Repeat 2 additional times, spin and aspirate supernatant as completely as possible before adding 50-60 ml of 1.5 or 2X LSB.

 

5.  Boil IP for 5'.  Load on desired gel.

 

 

 

 

*  If you are working with suspension cells, add 3-5 vol of ice-cold PBS, and spin at 5 krpm in tabletop centrifuge (4o C) for 3-5 minutes.  Pour supernatant off of pellet, and add lysis buffer to pellet; put pellet direclty into eppendorf and centrifuge.

 

**  In some cases, it may be desireable to spin out insoluble material in an ultracentrifuge (i.e. to remove lighter vesicles, get a cleaner IP, etc).  In this case, spin in Ti70.1 for 30-60 minutes at 55 krpm.