Hi,
I would like to visualize both poly-ubiquitinated protein species (from 63 kDa to 200 kDa) as well as free ubiquitin (8.5 kDa) on the same Western Blot. I’d be using a 4–15% Mini-PROTEAN® TGX™ Precast gel to separte the proteins and VU-1 (Life Sensors) antibody to detect them. Should I transfer these proteins using the same conditions or should I cut the gel into two pieces and use different transfer conditions, e.g longer and wet transfer for poly-ubiquitinated proteins and shorter semi-dry transfer for free ubiquitin?
Many thanks in advance,
Kati
you can use the same gel for one transfer, after revelation you will have many bands, and it will corssepond at the polyubiquitined proteins. a transfer for 35 minutes, on dry conditions, it will be ok. but to see the free unbiquitine, it will be hard, it's to small, and it can be out in 35 minutes, you can try a small tima if you dont have it in the same membrane.
Using a gradient gel ensures that both mono and poly Ub are transferred at the same efficiency. This is because small proteins like Ub "see" high % of gel while large proteins (such as polyubiquitylated conjugates) "see" low% of gel. Therefore, you ensure equal transfer by compensating for the differences in molecular weight by using a gradient gel. One more thing, for immunobloting you need to unfold Ub. However, adding SDS, DTT and boiling will not do the work. We use 8M urea in the sample buffer.
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