Faculty of Biological Sciences

Protein Identification

Useful for:

  • Confirming protein ID
  • Identifying unknown bands on SDS-PAGE gels
  • Determine interacting partners in pull downs
  • Localising post translational modifications
   
Protein ID

 

Samples for protein identification should be submitted in the form of either a 1D gel that has been Coomassie stained or in a suitable buffer solution. Please note that we cannot perform intact molecular mass measurments on samples supplied in gels, these must be prepared in solution or lyophilised, see here.

For gel samples, once the gel has been run, it should be handled inside a laminar flow hood where possible. This is to reduce contamination with keratin proteins that interfere with the identification. Rinsing all boxes, eppendorfs, gel plates, scalpels etc. that come into contact with the gel with 70 % ethanol can help to reduce the risk of keratin contamintaion.

Staining should be performed in clean gel boxes that have not been used for western blots. Where possible Expedeon InstantBlue Comassie stain should be used as this is MS compatible (n.b. gels stained with Generon Quick Comassie stain will NOT be accepted for analysis). Do not use excessively long staining times, bands should start to appear within 15 min depending upon the amount of material present. The MS Facility can provide aliquots of Expedeon InstantBlue stain for internal users using the service for the first time.

If you require ID of bands detected using silver staining, please contact the Faciltiy.

Please provide the entire gel to the Facility in water along with a photograph indicating which bands to excise for analysis. Please do not attempt to excise the bands yourself.

If you wish to submit a sample in solution please see below for the minimum sample amounts.

Minimum sample requirements

Gel Samples

The image below shows Coomassie-stained gel bands at different levels of protein loading:

To be suitable for protein ID analysis, there must be a clearly defined band as shown for each loading level in the picture above. Generally, the higher the loading on the gel the higher the sequence coverage acheived. The band appearence at different loading levels will vary protein to protein. Bands that are feint and/or diffuse, or require extreme settings during image acquisition to visulise are unlikely to give any sequencing results. If we need to tilt or hold a gel up to the light to visualise a band, it is not suitable for ananlysis and will not be run. Ideally between 1 - 5 µg protein should be loaded where possible.

In-solution samples

Single proteins should be between 0.1-1mg mL-1 concentration. For relatively simple protein mixtures (a few tens of proteins), 20-50 µg of total protein is required in volumes of between 50-100 µL. For complex protein mixutres (100+ proteins) at least 300 µg of protein should be submitted. Detergents, glycerol and very high salt concentration (0.2 M+) should be avoided where possible.

Sample submission deadlines

Protein ID is run on a weekly basis depending on demand. Processing begins each Tuesday and bands should be submitted no later than 9.30am on the Tuesday morning. Due to the demand on this service we are having to strictly enforce this deadline. Any samples submitted after this time will be held over until the following week.

Samples should be brought to the Mass Spec Facility in lab 9.107 of the Astbury building. You should download and complete a Protein ID sample submission form form to bring along with your sample. Results for Protein ID are usually available by the Tuesday following the start of sample processing.

Users external to the University can find additional information here.

Notes

When submitting samples, you should provide the name of the species used to express the protein as well as the expected species of the expressed protein. If the expected protein sequence differs from the wild type sequence in any way (e.g. inclusion of His tag or presence of TEV cleavage site) or the seqence is not present in the Uniprot database, please email the sequence in single letter amino acid format to mass-spec@leeds.ac.uk.