Hi, all,
I am trying to check if two proteins form a heterodimer and did native PAGE.. I cut the gel slice containing the band where the putative complex is and now want to do SDS-PAGE to resolve the 2 proteins. What's the procedure? I don't need to recover the proteins. Thanks for your help!
Hello Ana,
perhaps after revealing the heterodimeric protein with a PAGE, could be suitable blotting it on a nitrocellulose filter, with a proper buffer depending on the nature of the complex, submerging it in an envelop with a radioactive or fluorescent proof for a binding site.
You could electro-elute the protein from the native gel and re-run it on the SDS-PAGE gel. Sensitivity will probably be an issue, however, since the recovery will be low. Electro-elution methodology can be found here:
Article Cost-effective and efficient apparatus for electroelution of...
Equipment is available from Bio-Rad.
Thanks, @Antonio and @Adam.
Since I don't have any additional equipment (like the electroelution apparatus) I was just wondering if it's possible to cut the first gel and run it in the second? This is what this paper (Ghilarov(2017) Structure 25, 1549-1561) describes but without any detail. It is not possible to melt the gel (its polyacrylamide) ... So, he just fits the piece of the first (native) gel in the well of the second one?
I thought about that approach, but it seemed to me that the protein might elute from the gel too slowly and miss the stacking phase of the electrophoresis. If the native gel acrylamide percentage is quite low and the protein is not too big, it might work. Why not try it and see? You probably should soak the gel in SDS-PAGE sample buffer before putting it in the well. Also, the native gel should not have been fixed or stained after electrophoresis.
Another approach to this problem would be to use gel filtration chromatography instead of native PAGE as the first step.
- Badges
- Science method