High yield elution of proteins from sodium dodecyl sulfate-polyacrylamide gels at the low-picomole level. Application to N-terminal sequencing of a scarce protein and to in-solution biological activity analysis of on-gel renatured proteins.
A simple, reliable procedure for practically quantitative (90-98%) and fast (< 30 min) elution of proteins from SDS-PA gels is described with reproducible recoveries in the range from 100 to 1 pmol per band, which does not require the inclusion of detergents in the elution buffer. It consists in the combination of (1) highly sensitive on-gel protein detection (50 mol per band) with imidazole-SDS-zinc (reverse staining), (2) crushing of the protein band to produce 32-micron gel particles, and (3) vortexing of the slurry in a solution of a zinc-complexing agent, e.g. glycine 0.5 M or EDTA 100 mM (100 microliters for a 100-pmol BSA band), at room temperature. Eluted proteins can be directly analyzed by RP-HPLC, quantitatively loaded onto a PVDF membrane, or, provided that they are previously renatured on-gel, analyzed by biological activity tests. The application of the procedure to in-solution enrichment of scarce proteins for N-terminal analysis is shown.
Castellanos-Serra
L R
LR
Center for Genetic Engineering and Biotechnology, Havana, Cuba. protchem@serverdos.cigb.edu.cu
Fernandez-Patron
C
C
Hardy
E
E
Santana
H
H
Huerta
V
V
D000180:Acrylic Resins; D000595:Amino Acid Sequence; D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D008567:Membranes, Artificial; D008832:Microchemistry / Q000379:methods; D011145:Polyvinyls; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000502:physiology*; D012680:Sensitivity and Specificity; D017421:Sequence Analysis / Q000379:methods; D012967:Sodium Dodecyl Sulfate; D012996:Solutions; D013194:Staining and Labeling / Q000379:methods; D005465:Fluorocarbon Polymers
D000180:Acrylic Resins; D002021:Buffers; D003902:Detergents; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D012996:Solutions; C016680:polyacrylamide gels; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers