Protein aggregates SDS-PAGE
Posted 09 June 2010 - 08:56 AM
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.
Posted 09 June 2010 - 10:55 AM
Posted 09 June 2010 - 10:15 PM
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.
i second tat.. boiling will aid the reduction better... still if u have the aggregates.. that wud mean they are not disulphide linked!!
Posted 10 June 2010 - 06:30 AM
Posted 10 June 2010 - 07:03 AM
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).
Edited by K.B., 10 June 2010 - 10:32 AM.
Posted 10 June 2010 - 07:51 AM