SDS-PAGE % - what % gel should I use (Apr/18/2006 )

Hi all,
I am running a pilot expression study of a protein I am making using Invitrogen's pET 151-D TOPO. The protein including all the extras, like His tags etc. is 27kD. I ran an 8% gel ( I have not done this before). The gel contained samples of both induced and uninduced bacteria. The bacteria have a 25kD band (present in all cases), and the induced smaples did have a darker band in the ~25-~27kD range. What % gel should I run to resolve the two?
Any other tips would also be appreciated.

Thnak you,
Neil Stewart

-nmstew-

you might want to try a 10 or 12% gel.

-mdfenko-

for a better separation you could try up to 15%

-isis-

Greetings!

I think 15% would give you the best seperation at this range.

good luck

-monkeybaji-

QUOTE (nmstew @ Apr 19 2006, 05:39 AM)

Hi.

If you're looking to use the protein for some extra expts, why do you want to distinguish your protein from the exogenous E. coli protein(s) on a gel? The fact that there are E. coli proteins at about the same size isn't a crime, in fact, it gives you another nice lane in any purification gel you do (you know, "uninduced", "induced", "S/N post His-tag", "beads post His-tag" etc). Why not just purify it by the His-tag, and then repeat the gel?

-swanny-

This is just to make sure. The issue is that I suspect the darker band is my recombinant protein, but I want to seperate my 27kd band from the bacterial 25kd band. The bands rans so close on an 8% gel there is a possibility that the darker band could be bands running together, at which case I would have a false positive. I doubt that I do, but science being science I need evidence. A higher resolution gel will help me tell this. If it checks out I will scale up production to 500ml volumes and then HIs purify the recombinant. From there I plan on cutting off the unnecessary amino acids and running a gel of cleaned verses un cleand protein. Thank you to all who helped and gave advice.

-nmstew-