I have to separate two proteins of 86kDa and 80kDa respectively, however, I just cannot get a decent separation even in 6% polyacrylamide gel. To make matters worse, these two proteins are isoforms and there is no commercially available antibodies to differentiate either one. I am pretty sure raising an antibody against one of the isoform is not an option for me.

I am tying with the idea of reducing the gel to 4% (I am pretty sure I am pushing it already) or a 2D gel (both isoforms seem to have the same IP though).

  • 1
    Why are you reducing the gel % ? Increase the % for resolution. – WYSIWYG Dec 6 '14 at 7:10
up vote 1 down vote accepted

6kDa are usually enough to seperate two proteins in my experience. Try the other way, use a 7 or even 8% gel. They run longer but have a higher separation capacity. If you look at this figure from the LabFaQ, you see that you can use a higher percentage gel:

enter image description here

The other thing are the antibodies: Where are your isoforms different? 6kDa are 55 to 60 amino acids more for the long form, so there should be enough possibilities to make specific antibodies.

Your Answer

 
discard

By clicking "Post Your Answer", you acknowledge that you have read our updated terms of service, privacy policy and cookie policy, and that your continued use of the website is subject to these policies.

Not the answer you're looking for? Browse other questions tagged or ask your own question.