Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension.
We describe a modification in the sample application mode for isoelectric focusing with immobilized pH gradients. Instead of being applied at the surface of the gel in a sample cup, the sample is introduced into the gel during the immobilized pH gradient strip rehydration step. This modification implies the use of low percentage gels (below 3.5% T) and specially designed, but simple, rehydration chambers. The main advantages are a uniform resolution without side effects and the possibility of handling large sample volumes (500 microL for a standard 3 x 160 x 0.5 mm strip), allowing micropreparative work (milligram samples) with a simple experimental design.
Rabilloud
T
T
CEA-Laboratoire de Biologie, Moléculaire du Cycle Cellulaire, INSERM U309, Grenoble.
Valette
C
C
Lawrence
J J
JJ
D000818:Animals; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D013194:Staining and Labeling; D014867:Water / Q000737:chemistry
D011506:Proteins; D014867:Water