Non-specific antibody.

[Karin]

I have an antibody and western question. I have a really crappy antibody, but it's the only one made for my protein, and although I've figured out how to minimize background I still get multiple bands even in lanes with protein from cells. I know the height that my band should be and I know that my protein should be barely detectable in proliferating cells and detectable (with 100ug of concentrated protein) in differentiated cells and very detectable in heart muscle. My problem is I don't get consistent results looking at the band that I think is my protein. To check that the antibody is even able to detect the immunizing protein (IP) I tried running 1.7ug of IP and there was no band at the right height and multiple higher bands. I'm now running nanogram quantities of the IP to see if too much pure protein was the problem with that. Finally the QUESTION... has anyone ever worked with such a bad antibody and have any other suggestions of what I could try?

Thanks

[kmunson779]

Have you tried affinity purification of your antibody? You'll get a significant amount of loss, but it can really help if your antibody is that bad. For some protocols, try here.

[danfive]

What's the antibody, who makes it?

[danfive]

Also if WB is so bad try 2D gel separation followed by trypsinization and LC-MS-MS. The sequence LC-MS-MS will give you new sequences to synthesize (custom labeled peptides can then be used to quantitate sample digested peptides) and/or produce antibodies for.

[Karin]

Thanks for the advice. Abnova makes it and it's called Rrad. They have only tested it against the immunizing protein and I'm waiting to hear back from them about why the IP isn't properly detected.

[danfive]

Ras-related associated with diabetes (Rrad?) I checked it out in iHOP and NCBI Unigene EST expression profile. It appears that the EST expression profile matches exactly your original comment about tissue/develomental stage specific expression. So just in case you're interested in this non-WB approach I'll provide the links.

UNIGENE for Rrad ---> Ras-related associated with diabetes, Gallus gallus - UniGene Gga.12080
Scroll down this page and click Expression Profile (its under GENE Expression)

[danfive]

Here's a not too useful link to iHOP search results for RRAD.
---> iHOP - Information Hyperlinked over Proteins [ RRAD ]
It does provide links to all major info from NCBI, i.e.
NCBI Gene
NCBI RefSeq
NCBI UniGene
NCBI Accession