Co-Immunoprecipitations

Mikek kalch at ulam.generes.ca
Sun Jul 23 21:27:55 EST 1995


G'day...
I am attempting to IP a large protein using a monoclonal antibody and then
use Western blotting to detect a protein which should co-ip with my
protein of interest.  I have several questions for which I have read
several different answers to, and have heard a million different
suggestions from here on campus.
For more info... I am using a class IGg2a mAB, and using protein-A
sepharose beads to bring down the mAB.

1.  When I run my IP out on a SDS page I see two bands (as well as less
faint ones between 50 and 25 kDa), one at about 25 kDa and one at about 50
kDa, whats the scoop??  I know these are part of the mAb portions for I
don't see the bands in my lane with no mAb.

2.  When I wash my protein A-mAb mixture I am using 500 ul of my lysis
buffer with 1% NP-40 (and I am using about 250 ug of HEK293 cell lysate). 
I am afraid I may destroy the interaction of my co-IP protein under high
detergent or high salt conditions.  Any wash suggestions?

3.  On a tagent here, I have generated a different GST-fusion that codes
for a protein of about 50 kDa (no GST) and I am having a bitch of a time
trying to get this thing to come out of the bugs.  I have tried DH5 alpha,
BL21 and I am now onto UT5600.  Is there a good suggestion for this one
too?  (We are or should I say were a "DNA lab" but now I as a graduate
student am moving into uncharted territory for our lab, and I am getting
edgey? about things not working.)

3.  Any other suggestions regarding co-IPs or GST fusions would be much
appreciated.

Thanx

kalch



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