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-   -   Immonoprecipitation of ATXN2... unspecific band or heavy chain? (http://www.molecularstation.com/forum/immunoprecipitation-forum/84053-immonoprecipitation-atxn2-unspecific-band-heavy-chain.html)

kaaschr 11-16-2011 08:08 AM

Immonoprecipitation of ATXN2... unspecific band or heavy chain?
 
As a part of my master thesis i am working on immunoprecipitation of ATXN2 (145kDa) using an antibody from BD Biosciences (product 611378) raised in mice. The protocol i am using is from Nature protocols from Peritz et al (Immunoprecipitation of mRNA-protein complexes).

Following IP i run a western blot (Tris-Acetate buffer and a 3-8% agarose gel) using the same antibody for ATXN2 as primary antibody and Dako secondary antibody with HRP targeting mouse immunoglobulins.

I am a new user so i am not able to upload a picture of the Western blot and link to it but i can describe it as the following:
As controls i use a Myc-antibody raised in mice and the westen blot shows that only a heavy chain and a very weak light chain is seen. Another control is immonoprecipitation without and antibody and no band are visible.

When doing the IP using ATXN2 specific antibody I have used a larger concentration of antibody (20l = 5g) compared to the IP using Myc (5l = 1g) so the heavy and light chain bands are stronger on this lane compared to the myc. I am seeing a band corresponding to a light chain, one to the heavy chain, one to ATXN2... and one unspecific at approx 60 kDa right above the heavy chain. I have been unable to remove the band even with several extra washes and went online and found this forum. Anyone have an idea what the 60kDa band could be or what you would have done next if this was your IP experiment? Any suggestions/input are greatly appreciated.

kaaschr 11-20-2011 10:49 AM

Re: Immonoprecipitation of ATXN2... unspecific band or heavy chain?
 
Update: I did another atempt this week with a new protocol and when running the WB it loaded 500ng of ATXN2 antibody in one lane and found that bands came up at heavy (55kb), light chain (25kb) and the unspecific at 60kDa. It is a polyclonal antibody but is not all heavy chain 55kb? Anything other in the antibody mixture that could yield this band?

mmorgan 12-14-2011 11:48 AM

Re: Immonoprecipitation of ATXN2... unspecific band or heavy chain?
 
Quote:

Originally Posted by kaaschr (Post 437255)
Update: I did another atempt this week with a new protocol and when running the WB it loaded 500ng of ATXN2 antibody in one lane and found that bands came up at heavy (55kb), light chain (25kb) and the unspecific at 60kDa. It is a polyclonal antibody but is not all heavy chain 55kb? Anything other in the antibody mixture that could yield this band?

It's probably just a non-specific protein that is recognized by your antibody. If that's the only non-specific band you see consider yourself lucky to have quite a nice antibody! I wouldn't worry about it, do you have an ATXN2 negative cell line you can use as a control??


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