We have verified each step of in-gel digestion. We reduced the sample processing time and labor by optimizing the following four points.
1. Using intact gel pieces.
2. Use 96-well filter and positive pressure manifold before digestion.
3. Extract digested peptide without acetonitrile
4. In order to minimize non-specific absorption, digest in a low-peptides-binding microtube not in the 96-well filter or low-peptides-binding 96-well multiplate.