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My antibody recognizes two bands - I tried everything and I have still two bands (Jul/07/2005 )

I do western blot of spinal cord samples. Antibody antiparvalbumin detects two bands. First is parvalbumin around 12kDa. BUT it detects also band of 3 times higher intensity than that of parvalbumin 12kDa around 30kDa. I thought that it could be dimer, I tried all possible conditions of SDS-PAGE to destroy "dimer", unsuccesfully. Do you have any reports about this second band? Is it also parvalbumin or its a matter of monoclonal antibody, which recognize some sequence in other protein. I attached picture of this blot. Thank you very much.

-David Sojka-

Well, damn. I couldn't really see anything in the attachment.

But... I went thru this once for about 3 years (no kidding!) I knew I had the band, but it ran at 80kDa instead of 40kDa.

I'll tell you how I solved my problem.

Solubilize your protein in Lemmli's buffer... then add from a 1M stock of DTT (fresh made) so it is a final concentration of 100mM. I.E. if your volume of protein plus Lemmli is 20ul, add 2.2ul of 1M DTT. Let is sit at the bench for 30 min.

Then add (fresh made again) from a 500mM stock of Iodoacetimide (IAA) to a final concentration of 400mM to your sample. Yah, I know this increases the volume a lot.. but what can you do? I cannont get IAA into solution higher than 500mM.

Let it sit at room temp for 30 min. It most likely will turn your bromophenyl blue yellow... that is okay. When the 30 min are up, add 2-5ul of 1M tris pH 9.5 to your sample and the bob will turn blue again. Load on gel, run and blot as per usual.

Try it and let me know what happens.

Turned my 3 year old dimer into a monomer toot sweet.


Did you try to use a blocking peptide? Maybe it's useful to confirm that this second band really is your dimerized protein...