A procedure for protein elution from reverse-stained polyarcylamide gels applicable at the low picomole level: An alternative route to the preparation of low abundance proteins for microanalysis.
We developed a technique that allows rapid protein elution from polyacrylamide gel bands at room temperature into a detergent-free buffer (elution time 2 x 10 min, total working time about 30 min) with high yields (90-98%) even at a low picomole level (1 picomole per band). Its efficacy relies on the combination of protein detection by reverse staining with the enhancement of protein diffusion after gel crushing. Detection is accomplished by gel incubation in an imidazole solution, followed by incubation in a zinc salt solution to develop a negative stain pattern. Proteins are eluted by zinc complexation in Laemmli electrophoresis buffer (Tris + glycine), from which sodium dodecyl sulfate is omitted to allow direct subsequent microanalysis, e.g. high performance 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 sample reduction prior to electrophoresis on elution yields. For N-terminal sequencing, the protein eluate was automatically loaded on a polyvinylidene difluoride (PVDF) membrane with conventional HPLC equipment; both loading and membrane clean-up were monitored at 206 nm. By simultaneously processing several analytical bands, the procedure allowed trace enrichment of a natural scarce protein that was N-terminal sequenced.
Castellanos-Serra
L R
LR
Center for Genetic Engineering and Biotechnology, La Habana, Cuba. prot-chem@serverdos.cigb.edu.cu
Fernandez-Patron
C
C
Hardy
E
E
Huerta
V
V
D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004058:Diffusion; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D007457:Iodine Radioisotopes; D008832:Microchemistry; D010446:Peptide Fragments / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D017421:Sequence Analysis; D013194:Staining and Labeling
D001426:Bacterial Proteins; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D011506:Proteins