A Novel Protein Recovery Method via Gel Separation and Silica Columns Capable of Rapid Antigen Purification and Deep Proteomic Identification.

UNLABELLED: Gel electrophoresis is widely used for protein and nucleic acid analysis. Nucleic acid recovery from agarose gels is convenient and efficient, but protein recovery is not. This study aimed to develop and validate a novel method for recovering proteins from gels.

METHODS

After protein separation on gels, excised gel slices containing target proteins are processed using the same silica-column workflow as that used for nucleic acid purification, but with buffers specifically adapted for protein recovery. This approach only requires brief centrifugation steps and avoids laborious extraction and specialized equipment.

RESULTS

Proteome-wide recovery showed a slight preference of the method for low-molecular-mass and acidic proteins, while individual protein recovery efficiencies ranged from 69.9% to 86.7% and showed no correlation with intrinsic protein properties. The method effectively supported antigen purification. A banana (Musa acuminata AAA group 'Brazilian') proteome library constructed from recovered fractions contained over 18,000 protein groups and 27,000 individual proteins, which significantly enhanced protein identification in library-based data-independent acquisition (DIA) compared to directDIA analysis of single samples.

CONCLUSIONS

This method bridges gel-based protein separation- and column-based purification, making protein purification as simple, fast, and scalable as nucleic acid purification. It offers a practical alternative for proteomic sample preparation, antigen purification, and DIA workflows.