12CA5 MAb immunoprecipitation

[brett]

>Dear Netters,
>
>        We are trying to immunoprecipitate (from total yeast lysate) an 
>epitope tagged protein. The epitope is the HA tag YPYDVPDYA with the MAb 
>12CA5. We're having high background problems coming from non-specific 
>binding to protein A-sepharose beads. The antibody worked great for 
>western blot detection, but not at all for immunoprecipitation. My buffer 
>contains 50 mM Tris pH 7.5, 150 mM Nacl, 2 mM MgCl2, 1 mM EDTA, 0.5 % 
>NP-40 and 1 mM PMSF. 
>
>        Does anyone have a protocol for immunoprecipitation with the 
>12CA5 Ab ? Also, any suggestions, references or hints would be greatly 
>appreciated.
>
>
>Thanks in advance,
>
>J-Y Masson
>CHUL research center
>Quebec, CANADA

Your IP buffer is a little like glue. To decrease nonspecific binding, wash
3x in your buffer + 1mg/ml BSA (V), and 0.125% SDS (assuming this will be
compatible with your future use, such as SDS-PAGE), then once in TNE. This
should clean things up quite a bit.  7-)



Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain