University of Minnesota
University of Minnesota
College of Biological Sciences
http://www.cbs.umn.edu/

Protocols

 

University of Minnesota Customer Sample Submission:

Deliver samples to the lab for electronic sample log-in by core facility staff. Provide all sample preparation information and experimental goals. Complete electronic or hard copy version of the 'EFS Information Form;' email or deliver hardcopy form to core facility. Provide updated EFS numbers to the core facility when changes occur.

University of Minnesota Customer Required EFS Information Form

NOTE: All samples are analyzed in the order of receipt.

Sample Submission and Shipment Guidelines

1) CONTACT THE LAB

Call or send email to one of the core facility staff members regarding your project and request for services before you send samples to the core facility. In addition to detailed sample prep and experimental design details, provide total protein amount, cysteine alkylating reagent, species and amino acid modifications of interest (when applicable).

St. Paul Lab (phone 1): 612-624-7715
St. Paul Lab (phone 2): 612-625-2280

MPLS Lab (phone 1): 612-625-2279
MPLS Lab (phone 2): 612-624-2674

2) LABEL YOUR SAMPLE TUBES PROPERLY and COMPLETELY, as described here:

Use a thin Sharpie marker for tube labeling, as follows:

  • Top of tube: Your INITIALS
  • Side of tube
    • Date
    • Sample ID
    • Your last name

Place one layer of Scotch-brand (or clear) tape over the writing.
Do not use color lab tape.

3) MAILING ADDRESSES for non-UM sample submission

 

St. Paul Lab Mailing Address (Packages):
Center for MSP
University of Minnesota
43 Gortner Lab
1479 Gortner Avenue
St. Paul, Minnesota 55108

Minneapolis Lab Mailing Address (Packages):
Center for MSP
University of Minnesota
5-194 MCB
420 Washington Avenue
Minneapolis, Minnesota 55455

In-Gel Tryptic Digestion Protocol

Download the In Gel Trypsin Proteolysis Protocol

Protocol adapted from: Shevchenko, Andrej; Wilm, Matthias; Vorm, Ole; Mann, Matthias. Mass spectrometric sequencing of proteins from silver-stained polyacrylaminde gels, Analytical Chemistry, 68: 850-858 (1996).

For Review on gel-separated proteins see:

Patterson, S. D., Reudi Aebersold (1995) Mass spectrometric approaches for the identification of gel-separated proteins. Electrophoresis, 16: 1791-1814.

In-Solution Tryptic Digestion Protocol

Download the In Solution Trypsin Digestion Protocol

Protocol adapted from: Current Protocols in Protein Science, (1996-current) John Wiley and Sons, Inc. Brooklyn, Section 11.1.

Notes:

Read Promega's protocol for trypsin digestion (click on 'Protocol')

Stage Tip Protocol

Download the Stage Tip (Stop and Go Extraction Tip) protocol for C18 solid phase peptide extraction/desalting

ZipTip Protocol from MSP, adapted from Millipore

Download the ZipTip Protocol from MSP

Protocol can be accessed at Merk Millipore.

Coomassie (CBB-R 250) Destaining Protocol

  1. Cut band from the gel
  2. Wash several times with 10 mM dithiotreitol (DTT), 0.1 M 4-ethylmorpholine acetate (pH 8.1) in 50% acetonitrile (ACN); (3 minutes in microwave oven per wash is suggested). Repeat until the Coomassie is completely removed.
  3. Wash gel piece with water.
  4. Shrink with 100% ACN (5 min sonicate)
  5. Reswell with water (5 min sonicate)
  6. Shrink with 100% ACN (5 min sonicate)
  7. Reswell with water (5 min sonicate)
  8. Wash with ACN:H2O = 1:1 (5 min sonicate) (steps 3 - 7: salt and other contaminant removal)
  9. Speedvac to near dryness.
  10. Reconstitute sample with proteolytic digestion buffer→proteolytic digest

Reference: Karel Drbal, Pavla Angelisova, Ivan Hilgert, Jan Cerny, Petr Novak, and Vaclav Horejsi(2001) A proteolytically truncated form of free CD18, the common chain of leukocyte integrins, as a novel marker of activated myeloid cells. Blood, 98(5): 1561-6, with modifications by Petr Novak at the Laboratory of Molecular Structure Characterization, Institute of Microbiology, Prague

SYPRO (Bio-Rad) Ruby Protein Gel Stain Removal

  1. After staining with Ruby Gel Stain excise protein band/spot from gel
  2. Wash the gel slices 2 x 25 mM ammonium bicarbonate in 50% acetonitrile and 1 x 100% acetonitrile
  3. Dry slices using a Speed Vac
  4. Reconstitute sample with proteolytic digestion buffer

SYPRO destaining notes provided by Bio-Rad Laboratories Technical Services department (Aug 2001)