9 replies to this topic

[-Kay]

Hi, all!
I have been running blue Native PAGE gels using 3-12% bis-tris gels. I keep getting protein streaking, and no matter what I do, it won't go away. I have reduced the salt concentration of my samples, used the 5% G-250 coomassie sample additive, and spun the samples the remove any particulates, and reduced the amount of protein in my but the streaking endures. Any thoughts as to what could be causing it?

[mdfenko]

have you tried reducing the concentration of your samples (and protein load)? you may be seeing continuous aggregation of the protein.

do they streak all the way back to the origin or just a short way back? the streaking may be due to post translational modifications of the protein (glycosylation, lipidation, etc)

[-Kay]

Yes, they streak all the way back to the origin, and I've tried reducing the sample concentration.

I'm stumped

[mdfenko]

you can try to add a little 2-mercaptoethanol or dtt.

[-Kay]

how much would you recommend I add?

[mdfenko]

1-2mM dtt or 10mM 2-mercaptoethanol (to start, you may need to go up to 100mM) may prevent aggregation.

[-Kay]

Thanks so much! I'll try that out and see how it works out

[-Kay]

Sorry, I had a slow moment... Won't adding b-mercaptoethanol denature my proteins, making it pointless for a NativePAGE gel?

I'm trying to determine the size of my proteins in their native state. Since gel filtration didn't work, my next option was to try this Novex NativePAGE gel system (charges the proteins with coomassie). Because of the streaking, it hasn't been helpful either.

[mdfenko]

-Kay, on 15 February 2011 - 07:22 AM, said:

Sorry, I had a slow moment... Won't adding b-mercaptoethanol denature my proteins, making it pointless for a NativePAGE gel?

low amounts of dtt or b-me will prevent disulfide bonds from forming (protect free sulfhydryls) but will not break existing bonds. they may prevent aggregate formation without denaturing the protein.

[-Kay]

I understand. Thanks!!