What do I need to do to submit a sample for a single protein band?
Contact us for an initial consultation at pmaf@austin.utexas.edu
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
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.
Take a photo of the gel with sample bands and marker lanes visible.
Cut out the band with a clean scalpel, trimming excess gel. Excess gel will be detrimental to the analysis.
Cut the gel band into 1 mm cubes following the tutorial below:
Place the 1 mm cubes into a 1.5 ml Eppendorf tube pre-rinsed with 50% organic solvent (MeOH or ACN) and milliQ/ultrapure water and cover them with 500 uL of destain solution.
If you submit bands from multiple lanes as one sample, cut out and discard the empty space between the lanes and chop each band into 1 mm3 pieces.
For all samples:
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. The work order will generate a four-digit PF number.
Submit the gel photo as an attachment on the sample submission form.
Gel bands/plugs must be chopped into 1 mm cubes by the user prior to submission.
Each sample needs to be labeled using the PF number. Label the caps of the tubes sequentially using the order number and sample number (PF#-1, PF#-2, 3......).
Bring them to MBB 1.420 or ship them to Biological Mass Spectrometry Facility, MBB 1.420, 2500 Speedway Ave, Austin, TX 78712.