Trouble with IEF gels - (May/09/2005 )
I've recently started a project using IEF gels from Invitrogen.
My trouble is that, though the IEF marker focalises well, poor focalisation occurs with my samples and it seems that most of the sample is lost (Coomassie staining, after fix in TCA as recommended), compared to similar loads on SDS PAGE gels.
Better results could be obtained using Ampholine PAGPlate from Amersham, but I'd prefer to go on with Invitrogen equipment, for economical reasons !
I do work on yeast samples, either prepared in Tris 50mM (no salt !) or Tris 50mM plus Urea 8M.
Has anyone of you worked with Novex IEF gels ? Any advice to give on how the samples should be prepared, on how much sample should be loaded ? ...
It seems lackof focusing is due to salts in the TCA proteins sample. I would suggest that after TCA precipitation, dialyze again to remove any residual salts in the sample or use desalting columns (I suggest this strongly). Good Luck!
I had good results in washing the pellet from acetone precipitation two times with 70%EtOH to desalt the sample. In the SDS-PAGE I got much better separation.