Protein Enzymatic Digestion/Chemical Cleavage

In Gel Digestion

Proteins of interest can be submitted for proteomic analysis (i.e. protein identification) as Coomassie Blue (R250, G250 or "colloidal"), Cy dyes, SYPRO® Ruby or digestion compatible silver stained 1D and 2D gel bands. Trypsin is the default enzyme, but by request, a variety of other enzymes (e.g., chymotrypsin, endoproteinase GluC, etc.) may also be used. Digestions can also be performed on samples that have been blotted onto PVDF membranes, but we strongly encourage investigators to avoid this unnecessary step and instead, submit the SDS PAGE gel band or spot for analysis. In gel robotic tryptic digestion is also available with a minimum of 24 protein bands or spots for a reduced rate. Please note that for silver stained protein gel samples, the silver will be removed for an additional fee. Typically, a visible band (except for the Cy dyes) contains sufficient material for identification of the protein.

In Solution Digestion

Samples that do not require SDS PAGE purification or for MudPIT analysis, may be submitted dry (after vacuum centrifugation in a Speedvac) in 1.5 ml Eppendorf tubes that do not contain O-rings. Although these samples generally may contain up to the equivalent of 50 μl of 0.5 M non-volatile salts, the investigator should email the details of these samples (i.e., exact buffer composition and estimated protein concentration) to the MS & Proteomics Resource prior to final sample preparation. Unless requested otherwise, the Keck Laboratory will reduce, carboxamidomethylate and then digest these samples with trypsin (or a dual digestion of Lys-C followed by trypsin for complex samples) in a final concentration of 2 M urea, 50mM ammonium bicarbonate. In addition to utilizing other proteolytic enzymes, samples that are submitted for in solution digests may also be subjected to cyanogen bromide cleavage upon request. Recommended sample amount varies depending on the on the type of proteomics experiment.