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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

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Abstract

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-μm 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 μl 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.

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Authors and Affiliations

  1. Center for Genetic Engineering and Biotechnology, Havana, Cuba

    Lila R. Castellanos-Serra, Carlos Fernandez-Patron, Eugenio Hardy, Hector Santana & Vivian Huerta

Authors
  1. Lila R. Castellanos-Serra
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  2. Carlos Fernandez-Patron
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  3. Eugenio Hardy
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  4. Hector Santana
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  5. Vivian Huerta
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Castellanos-Serra, L.R., Fernandez-Patron, C., Hardy, E. et al. 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. J Protein Chem 16, 415–419 (1997). https://doi.org/10.1023/A:1026340923032

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  • DOI: https://doi.org/10.1023/A:1026340923032

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