samples spread after loading - (Feb/25/2008 )
hi!
I´m having problems with mi SDS-PAGE gels for WB. I make a 10% resolving gel and a 6% stacking gel, they seem to polymerize fine and wells look nice. Then I load my sample as always but almost in all wells in a few minutes the sample leaks and mixes with the sample of the next well (it doesn´t bubble out, it seems to go through the bottom of one well to the next one 
) and also spreads downstream all over the stacking gel.
Any idea about what´s happening? 
I´ll try to post a pic. Thank you.
It sounds as if you perhaps use combs which are too big compared to the spacers. Is everything of the same width? Or do you put your pipet tip too deep in the pocket so that the there is room between the gel and the glass plates? Are the screws put on tightly?
Thanks biomaus. I use the Biorad Miniprotean 3 system, the spacers are sticked to the bigger glass and I use the combs provided with the glasses, 0.75 mm. I have been using this system for more than 1 year without problem
There are no screws, just fit it properly. About the tip...it´s a good idea but I don´t know if that could be the reason because I load as always and I hadn´t this problem before
but anyway I´ll try putting the tip less deep and let the sample slide from glass to the bottom of the well. Hi, you could try to wash glasses very well with alchol, and dry, before set up electroforesis machinery. Sometime happens that polyacrilamide doesn't adhere onto glass because it is dirty..
S.
S.
I second this. It sounds as though your acrylamide isn't sticking to the glass, but a thorough cleaning should nick that problem in the bud.
Ginger
S.
I second this. It sounds as though your acrylamide isn't sticking to the glass, but a thorough cleaning should nick that problem in the bud.
Ginger
Thank you guys, I´ll clean better my glasses and see what happens.