A PROCEDURE FOR PROTEIN ELUTION FROM REVERSE-STAINED POLYACRYLAMIDE GELS APPLICABLE AT THE LOW PICOMOLE LEVEL - AN ALTERNATIVE ROUTE TO THEPREPARATION OF LOW ABUNDANCE PROTEINS FOR MICROANALYSIS

We developed a technique that allows rapid protein elution from polyac rylamide gel bands at room temperature into a detergent-free buffer (e lution time 2 X 10 min, total working time about 30 min) with high yie lds (90-98%) even at a low picomole level (1 picamole per band). Its e fficacy relies on the combination of protein detection by reverse stai ning with the enhancement of protein diffusion after gel crushing, Det ection is accomplished by gel incubation in an imidazole solution, fol lowed by incubation in-a zinc salt solution to develop a negative stai n pattern. Proteins are eluted by zinc complexation in Laemmli electro phoresis buffer (Tris + glycine), from which sodium dodecyl sulfate is omitted to allow direct subsequent microanalysis, e.g. high performan ce liquid chromatography (HPLC) and automatic Sequencing, A variety of proteins were eluted efficiently (with no apparent restriction due to their intrinsic properties) as quantified with radioiodinated total E . coli proteins. Yields were independent of acrylamide concentration, protein molecular mass (from 10 to 100 kDa) and the amount (from 1 to 100 picomole) of protein in the band. This protocol was derived from a quantitative evaluation of the effect of protein staining and of samp le reduction prior to electrophoresis on elution yields. For N-termina l sequencing, the protein eluate was automatically loaded on a polyvin ylidene difluoride (PVDF) membrane with conventional HPLC equipment; b oth loading and membrane clean-up were monitored at 206 nm. By simulta neously processing several analytical bands, the procedure allowed tra ce enrichment of a natural scarce protein that was N-terminal sequence d.