proteomic study at 1-D and 2-D electrophoresis. - wants your opinion ! (Nov/27/2006 )
i am studying the chemopreventive effect of one NSAID on one cell line. my drug has very good result at the antiproliferation activity. i am intersted in studying the protein expression after treatment and control.
so first i want to see the difference in the protein band on 1-D electrophoresis. as i am new to protein biochemistry. so it is becoming very difficult for me run a very good gel for the camparision purpose.
i will be very happy if some other also working on the same strategy.
my protein band are usually concentrated on only upper part of gel. i run 12% gel.
is there any site for troubleshooting the SDS-PAGE.
kindly help me in this direction.
your help will be highly appreciated.
Try to make a search in google (www.google.com). Many companies publish "trubleshooting guide".
As regard the band concentration in the upper side of the gel, try to run more the gel.
i run my gel untill the dye come out from the gel, but still the bands come within half the length of the gel.
you have to make your gel thinking about the molecular size of your protein or proteins mixture. Perhaps they are too bigger for a 12% gel and so you would have to try with a lower acrilamide concentration so bigger proteins can migrate more.
if your are dependent on prices reproducibility use precast gels; to find differences in protein expression you will not circumvent to separate proteins in 2D; as a beginner try complete systems
1) Which is the MW of your protein? If you tell us we can try and understand which %polyacrylamide you should run.
2) Are you quantifying the protein extract? Are you sure you are loading exactly the same amount of extract in the control and in the treated cells?