Boiling IP samples - (Aug/07/2008 )

Hello,
I completed an IP yesterday and boiled the samples at 95*C for 5 min. I then froze the samples at -20*C overnight. I will run the samples today. My question is: should I boil the samples again this morning before loading my gel?

Thanks!

One additional question: Why do some people heat their samples to 65*C instead of 95-100*C?

-jlolsen-

[quote name='jlolsen' date='Aug 7 2008, 12:07 PM' post='146398']
Hello,
I completed an IP yesterday and boiled the samples at 95*C for 5 min. I then froze the samples at -20*C overnight. I will run the samples today. My question is: should I boil the samples again this morning before loading my gel?

Thanks!

I've boiled my samples the night before then ran them on a gel without re-boiling them. It shouldn't be a problem.

as for the second question, no idea.

-labrat612-

You should not reboil your sample. It may result in protein degradation.

The same answer applies to the second question. Boiling at 65 C may help protect heat-sensitive proteins from degradation, although it can also lead to incomplete denaturation.

Hope this helps.

-Madrius-

as madrius says, some use 65C to prevent protein aggregation that may occur to some proteins with prolonged exposure to 95-100C.

65C for 10-20 minutes should give complete denaturation.

-mdfenko-

QUOTE (mdfenko @ Oct 24 2008, 12:37 PM)

as madrius says, some use 65C to prevent protein aggregation that may occur to some proteins with prolonged exposure to 95-100C.

65C for 10-20 minutes should give complete denaturation.

thanks, didn't know I could boil at 65c instead of 95c.....so next time when I do IP I'll boil the beads in SDS-PAGE buffer at 65c for 20mins to remove all antibodies/proteins!...cuz I'm not a fan of 95c-100c at all....I'm scared low molecular weight proteins would degrade at that temperature.

-Curtis-