In my experiment, I need to do 2-D gel electrophoresis of Paca2 pancreatic cancer cell line. But after coomassie blue staining, there are always horizontal streaks. I think it's the problem with cell lysate or first dimension seperation. For cell lysate, I use TNN buffer (50mM Tris(pH7.4), 150mM NaCl, 20mM EDTA(pH8.0), 50mM NaF, 0.5% NP-40, and 1mM Na3VO4), supplemented with 2mM PMSF and 1% DTT. For 2-D procedure, I follow the instruction of ReadyPrep 2-D Starter Kit(Bio-Rad), so the first dimention seperation is: 250v 20min linear, 8000V 2.5hr Linear, 8000V 25000V-hr rapid. I use pH3-10 IPG strips 11cm. Could anyone familar with 2-D give me some suggestions about how to improve and get rid of horizontal streaks?
Thank you in advance,
2-D gel electrophoresis of Paca2 Cells
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