App Note

In-Gel Digestion Procedure for Protein Sequencing

In-gel digestion simplifies sequencing-based protein identification workflow

Complex mixtures of proteins complicate proteomic research workflows. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is commonly used to separate these mixtures, but requires additional subsequent procedures for protein identification. One of the most prevalent protein identification techniques post-SDS-PAGE separation is Western blot. However, the antibody-based method is impacted by the availability of validated commercial antibodies and an inability to differentiate subtle amino acid sequence variations or isoforms, especially when protein sizes are similar. Antibody-related challenges often lead to wasted time and resources or erroneous results. 

In-gel digestion simplifies sequencing-based protein identification workflow

New next-generation sequencing technology provides a promising alternative to antibody-based methods of protein identification. This technical note presents a novel in-gel digestion procedure compatible with the downstream library preparation and sequencing workflows that combines the benefits of gel protein separation with next generation sequencing.

This technical note introduces an in-gel digestion technique as an alternative to antibody-based methods, offering a more sensitive approach to protein identification. The process involves the extraction and sequencing of peptides from proteins resolved on SDS-PAGE gels, bypassing the constraints of antibody specificity and availability. This methodology not only refines the precision of protein identification but also aids in discerning between closely related proteins or isoforms, a critical advantage in understanding complex biological systems.

Download the technical note for a detailed workflow for protein sequencing library preparation from gels, exploring the potential for separating and enriching complex sample mixtures to resolve differences in amino acid sequence.

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