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In-gel protease digestion service

eagle-i ID

http://eagle-i.itmat.upenn.edu/i/0000013f-67fa-3f36-a468-831a80000000

Resource Type

  1. Material modification service

Properties

  1. Fee for service
    Yes
  2. Resource Description
    "Purpose: Enzymatic digestion of proteins in polyacrylamide gels is the preliminary procedure for multiple alternative services including LC-MS/MS, Gel/LC-MS/MS, Peptide mapping, and PTM analysis. The micro digest used here has been developed to minimize volumes and maximize peptide yields which are typically >80%. Porcine-modified trypsin, the protease of choice, cleaves at the carboxyl side of lysine and arginine residues. For special applications such as analysis of PTMs, other proteases also can be used. Requirements: Submit an intact 1D or 2D gel stained with colloidal Coomassie® (preferred) or MS-compatible silver stain that was prepared following our "Sequence-quality SDS-gel guidelines." For simple protein identification using LC-MS/MS, as little as 5 femtomoles of the desired protein in a single mini-gel band or 2D gel spot typically will yield identification of at least the most abundant protein present; that is, even barely detectable silver stained bands will result in identifications, although sequence coverage will be quite limited when near the limit of detection. After staining/destaining gel, rinse it for one hour with high purity water, seal in plastic bag, photograph or scan the gel, and mark the desired protein band(s) on the gel image (annotated image in PowerPoint preferred). Store gel at 4°C until the entire gel is delivered to the facility. Results: Small-volume tryptic digest of purified protein or complex protein mixtures for further characterization via LC-MS/MS, etc. Purified proteins invariably can be identified from barely detectable Colloidal Coomassie® bands/spots, and most barely detectable silver-stained bands/spots usually can be identified with limited sequence coverage. Gel slices with intense Colloidal Coomassie® staining from complex proteomes can yield confident identification of >100 proteins for each slice, and up to several thousand proteins from an entire gel lane. "
  3. Service Fee URL
    http://www.wistar.org/our-science/shared-facilities/proteomics-facility/user-fees
  4. Service Provided by
    Proteomics Facility (Wistar)
  5. Website(s)
    http://www.wistar.org/our-science/shared-facilities/proteomics-facility/services
  6. Related Technique
    Trypsination
 
RDFRDF
 
Provenance Metadata About This Resource Record
  1. workflow state
    Published
  2. contributor
    fcoldren
  3. created
    2013-06-21T14:34:27.722-04:00
  4. creator
    fcoldren
  5. modified
    2013-07-02T10:59:05.872-04:00

Copyright © 2016 by the President and Fellows of Harvard College
The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016