HPV E7 protein - (Nov/06/2007 )
do you have any suggestions how to concentrate my gel (i would suggest 15% polyacrilamide gel)?
The size of E7 protein is ~18kDa if i mention right.
I found some paper showing endogenous E7 protein in HeLa cell lysates, so i started to read several other paper to get familar with E7 WB analysis.
Mostly, E7 protein was directly fixed with Glutaraldehyd on NC-membrane after blotting to prevent loss of E7 protein by washing the NC-membrane. I will try this out.
I dont know exactly how long i should run the gel. And of course, i have no idea weather i should scale down the blotting time.
Normally the lab use WB analysis for larger proteins ~100kDa.
Here some facts of our common used WB-protocol
- 12% running gel
- gel running time <90min
- blotting time 90min
My alterations might be:
- 15% running gel
- gel running time 60min to get stronger bands?
- 45min blotting time?
Would be nice to profit by your experience...
While I would highly recommend the 15% gel, you don't really need to bring the running time down. Just keep an eye on your dye front while running the gel and you should load enough marker so that you can follow where your size of interest is. Just don't let it run off the bottom. I'm not sure what you mean by "concentrate" your gel. You don't loose protein as the gel runs. If you are having trouble detecting you may just need to load more protein into the next gel. As for the blotting, you should be ok at 90min (I use about 70V) but you may want to use a smaller pore nitrocellulose or PVDF. 0.4um is typical but there is 0.2um available and this smaller pore membrane is nearly impossible to over-blot.