An efficient in‐gel digestion method on small amounts of protein sample from large intact gel pieces

Abstract In‐gel digestion is an essential method, but there has been concern that a large ratio of gel volume to protein volume will reduce digestion efficiency and make it difficult to recover peptides after digestion. Few methods for handling large gel pieces have been established. Therefore, we optimized the in‐gel digestion method to handle large intact gel pieces containing small amounts of proteins. As a model for large intact gel pieces containing small amounts of protein, gel pieces of 7 × 10 mm in size obtained by short‐range sodium dodecyl‐sulfate polyacrylamide gel electrophoresis of 150 ng of total protein extracted from Arabidopsis cells were used. The use of a positive pressure manifold and 96‐well filter enabled rapid and uniform liquid exchange and gel drying, even when handling multiple gel pieces, and reduced the labor required for a liquid exchange. This facilitated the study of conditions using multiple gels and enabled a detailed study of the enzyme solution permeation and elution methods. By using the optimized method, we were able to establish a method for recovering small amounts of protein from large intact gel pieces without compromising reproducibility or recovery rate.