A quantitative investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure.

摘要: We report the results of a systematic investigation to quantify losses protein during well-established two-dimensional polyacrylamide gel electrophoresis (2-DE) procedure. Radioactively labelled proteins ([ 14 C]bovine serum albumin and homogenate prepared from liver rat that had been injected with [ 35 S]methionine) were used, recovery was quantified by digesting pieces in H 2 O subjecting digests liquid scintillation counting. When samples loaded onto first dimension immobilised pH gradient strips in-gel rehydration, 44-80% amount loaded, depending on sample. Most unrecovered appeared have adhered reswelling tray. Losses isoelectric focusing (IEF) much smaller (7-14%), although approximately 2% migrate sample adjacent blank focussing apparatus. A further 17-24% lost into buffers equilibration prior running second dimension. run subsequent staining SYPRO Ruby amounted less than 10%. The overall loss 2-DE reduced 25% when IEF using cups instead rehydration. These extensive variable procedure mean spot intensities gels cannot be used derive reliable, quantitative information amounts present original