Protein aggregates SDS-PAGE - (Jun/09/2010 )
I was wondering if someone could help me please. I have been having a problem of having protein aggregates at the top of my gel when I run SDS-PAGE. I run 20ug of protein at 150V for 1 1/2 to 2 hours (7.5% Tris-HCl gel from BioRad). Before loading the gel, I incubate my samples in a 1:1 ratio in LSB for 30 minutes at room temperature. This past time I have increased the incubation time to 1 hour at room temperature and had the same problem. We use LSB from BioRad (cat.# 161-0737) and the recipe in my LSB is as follows:
62.5mM Tris-HCl, pH 6.8
0.01% bromophenol blue
And I add 5% b-mercap.
I was considering incubating my sample + sample buffer at 60C for 5-10 minutes instead. Does anyone think this would help my problem? All help is appreciated. Thanks.
Incubation at RT is a bit strange if you ask me, unless you have a specific reason to do that. Usual protocol is to boil samples for 5 minutes. My friend is also centrifuging her samples after boiling in order to remove particles (eg. aggregates) before loading.
K.B. on Jun 10 2010, 12:25 AM said:
i second tat.. boiling will aid the reduction better... still if u have the aggregates.. that wud mean they are not disulphide linked!!
Thank you both for your replies! I will try boiling my samples next time and see if they are properly reduced. Would you say 65C for 5-10 minutes is sufficient? Also, a couple other questions. Do you put the samples back on ice after boiling to stop the heat? And when your friend spins her samples down, at what speed?
As good as I know you need >70 deg.C to cleave disulphide bonds.
I don't put my samples back on ice after boiling with sample buffer.
My friend is centrifuging samples AFTER boiling with sample buffer, BEFORE loading them on the gel, approx. at 10000xg for 1 minute (at RT).
Great, thank you for the information! I will give this a shot and hope it fixes my problem.