Verify that the protein sequences of the species under study is in the Uniprot database. If not, you will have to supply a FASTA formatted protein sequence database electronically to our core at the time of sample submission at pmaf@austin.utexas.edu
For a single protein band:
Run a mini-gel and stain with Coomassie. Sypro ruby can also be used, but silver stain bands may not contain enough protein for identification. Estimate the amount of protein in your band by comparison to a marker of known concentration. DO NOT stain for more than 15 minutes.You only want to visualize the area of the plug. If you stain the gel too long, dye debris causes problems during the desalting step. Cut out the band with a clean scalpel, trimming excess gel. Excess gel will be detrimental to the analysis. If you submit bands from multiple lanes as one sample, cut out and discard the empty space between the lanes!
For a complex sample (e.g. lysate):
Do not run the gel to completion; only run the sample no more than 0.75 cm into the running gel (if a stacking gel is present). For precast gels without a distinct stacking gel, just run the sample no more than 0.75 cm out of the well. The plug should be no wider than a single mini-gel lane (~2mm). It is helpful to include a kaleidoscope marker to verify that the high molecular weight proteins have entered the gel before stopping the run. Stain the gel as above. Do not include the dye front in your gel plug, as the samples will not run properly on the LC. Cut out only the Coomassie stained protein area in the gel plug. Please also provide us with an estimate of the amount of protein loaded on the gel. The left lane shows the marker proteins run ~5 mm out of the well. The goal is to run the proteins into the gel without letting them separate, to put all the proteins into the smallest amount of gel possible. Ideally, the gel plug is ~ 2mm wide. If you submit a gel plug >5mm in height x 7.5 mm width, we will bill for additional digest charge for each 5 mm of gel. Also, you will have fewer protein IDs if you have excess gel. Trim all excess gel before submission.
Put each gel band or plug in a separate eppendorf tube with 500 ul destain.
Fill out a work order for Protein ID on FBS https://fbs.research.utexas.edu/Anon/Logon.aspx Contact pmaf@austin.utexas.edu if you do not have a log-in yet.
Put your order number on the tubes and sequentially number them (1,2,3......) Bring them to MBB 1.420 or ship them to Proteomics Facility, MBB 1.420, 2500 Speedway Ave, Austin, TX 78712.
