co-immunoprecipitation problem

[Danny Altshuler]

bg2z at musicb.mcgill.ca wrote:
: I am using a rabbit antibody to immunoprecipitate a protein under 
: co-immunoprecipitation conditions, which should, and does bring down a complex 
: containing another protein (HA tagged) against which I have a mouse Ab 
: (12CA5). The problem I have is that my initial immunoprecipitating rabbit Ab 
: is recognized on the nitrocellulose by the secondary Ab I use to detect 12CA5 
: (Goat anti mouse HRP- I detect using ECL) and the heavy chain migrates almost 
: exactly where the tagged protein is located. I know my protein is there 
: because there is always extra signal right above the IgG heavy chain, but it 
: would look much nicer if there was a way to degrade, or get rid of 
: specifically the IgG heavy chain. Other than running really loose gels (7% and 
: less) is there a way to get rid of that fat whopping IgG band? any advice will 
: be greatly appreciated

: Dino De Angelis, McGill Biochemistry
Dino, what I usually do is after the IP resuspend in Laemli buffer 
whitout DTT or b-MSH. So all the IgG (>90%) will run as a 150K protein.
Try to minimize the amount of primary antibody you use for the IP.
HOPE THIS HELPS
DANNY