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).

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    $\begingroup$ Why are you reducing the gel % ? Increase the % for resolution. $\endgroup$ – WYSIWYG Dec 6 '14 at 7:10

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.


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