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bioRxiv · 10.1101/2023.06.15.545122

Centrifugal Gel Crushing for Gel-based Proteome Analysis

Nishida, H.·Kanao, E.·Ishihama, Y.
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Abstract

We have developed a centrifugal gel crushing method using a pipette tip. Polyacrylamide gel slices are extruded from the narrowing cavity of a pipette tip by centrifugation in a few minutes to crush them into pieces of appropriate size. The size of the crushed gel could be controlled by several parameters, including centrifugal force and pipette tip cavity. In shotgun proteomics, gel-based LC/MS/MS, so-called GeLC/MS/MS, involves the essential but tedious processes of pre-fractionation by SDS-PAGE, followed by dicing the entire gel lane into several parts, fine dicing, and in-gel digestion after the diced gel is manually transferred to a microtube. In this study, we developed an alternative way to crush the pre-fractionated gel slice into small and irregular-shaped gels by centrifugal extrusion of the sliced gel from the narrow cavity of a pipette tip. As a result, we observed an improved recovery and reproducibility of digested proteins compared to the conventional method of manual dicing. We believe that this simple and rapid method of crushing polyacrylamide gels, which allows for parallel operations and automation, is useful for GeLC/MS/MS analysis and applicable to other approaches including top-down proteomics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/545122v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@da02dcorg.highwire.dtl.DTLVardef@f1dee1org.highwire.dtl.DTLVardef@15fb780org.highwire.dtl.DTLVardef@10848b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Nishida, H., Kanao, E., Ishihama, Y.. 2023-06-15. Centrifugal Gel Crushing for Gel-based Proteome Analysis. https://doi.org/10.1101/2023.06.15.545122

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