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We recommend conventional Coomassie or Invitrogen Colloidal Blue (P/N LC6025) (Coomassie) stain. We do not recommend silver-stained gels at present. If you
use conventional Coomassie stain, stain and destain the gel with <10% acetic acid. Higher concentrations will interfere with digestion.
Excise protein bands/spots of interest from the stained polyacrylamide gel. Use a clean scalpel, and cut out the darkest part of the band, leaving the light edges behind.
To avoid or reduce contamination with human keratins, cleanliness is very important throughout the procedure. Be very careful to avoid dust particles, always
wear gloves and preferably work in a laminar-flow hood.
Excised bands can be placed in Eppendorf tubes and shipped without refrigeration if sent by an overnight service. Use Parafilm to ensure that the tubes don’t come open during shipment.
The efficiency of protein digestion and peptide extraction depends on properties of the specific protein (degree of hydrophobicity, pI, etc.). In general,
picomole levels are preferred to lower levels, and more is almost always better; however, more gel pieces is not necessarily better. Overall efficiency is much
higher with a single concentrated gel band or spot than with several more dilute gel pieces.