The following protocols are routinely performed in the LSUHSC Proteomics Core. This information is provided for those clients who wish to perform their own electrophoresis and staining. Core staff are available to assist in design and implementation of client initiated experimentation.
2-D Gel electrophoresis
This technique separates protein via two discrete steps. The first step is Isoelectric Focusing which separates proteins based on their charge. This first dimension is then embedded onto an SDS-PAGE. The second step separates proteins based on size. This process can be enhanced for quantitative data by incorporating the use of DIGE (difference in-gel electrophoresis) system from GE Healthcare.
Some useful links on 2D electrophoresis and DIGE:
- Swiss Institute of Bioinformatics - http://world-2dpage.expasy.org/swiss-2dpage/docs/protocols/
- Bio-Rad - http://www.bio-rad.com/en-us/life-science-research/applications-technologies
- GE Healthcare - http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-US/products/2-d-electrophoresis-and-2-d-dige
The Core Facility recommends Sypro Ruby and Colloidal Coomassie (Invitrogen or Bio-Rad) staining methods. Both staining methods utilize non-hazardous components that offer superior sensitivity to other staining methods. These stains also preserve the proteins for subsequent mass spectroscopy identification.
Protein identification via mass spectroscopy is achieved through trypsinization of proteins. Partial sequence information is obtained from the mass spec and searched against an in silico trypsinized protein database. This is a very sensitive process and detection limits can be as low as femtomolar. Therefore, it is important to avoid contamination from other proteins. The major contaminants from gel samples are human keratins from skin and hair. To reduce keratin contamination use clean equipment and a fresh pair of gloves right before handling all gels. Also, try to grab the edge of the gel rather than the areas where the protein bands or spots are during the staining and rinsing steps. Keep gels covered to minimize the exposure to dust.
In-Gel Destaining and Digestion
Prior to trypsin digestion, the gel plugs have to be destained and equilibrated with buffer. The Core has a robotic system that can automate this process.
Destaining Gel Plugs (for Coomassie and Sypro stains)
- Working in a dust- free area, pick gel plugs for the protein of interest.
- For one-dimensional gels, further slice the bands into 0.5-1 mm3 cubes (~0.75-1 mm each side). Place gel plugs in clear polypropylene tubes or 96-well plates. For 2D gels pick as much of the spot without collecting too much background acrylamide.
- Incubate gel plugs with agitation in 100 µl 50 mM ammonium bicarbonate in 50 % methanol for 20 minutes. Remove the wash solution and repeat once more with fresh destain.
- Incubate gel plugs with 100 µl 75% acetonitrile for 20 minutes. Remove acetonitrile solution and dry gel plugs for 10 minutes.
In-gel Trypsin Digestion
- Prepare 20 µg/ml trypsin by adding 1ml of 20 mM ammonium bicarbonate to a vial of lyophilized trypsin.
- Add 8-20 µl trypsin solution to each tube to cover the gel plugs. Seal plate or tube.
- Incubate gel plugs at 37oC overnight.
- Add 50 µl 50% acetonitrile/0.1% TFA. Incubate and agitate at room temperature for 20 minutes. Remove supernatant and save.
- Add 50 µl 50% acetonitrile/0.1% TFA. Incubate and agitate at room temperature for 20 minutes. Remove supernatant and combine with earlier saved extract.
- Dry combined extract in a speed vac. This dried sample is ready to be submitted for mass spectroscopy identification.
Depending on the size of 1D gel bands, the volume of trypsin and extraction can be adjusted accordingly.
The Proteomics Core Facility is located in the Clinical Science Research Building room 331. For further information please contact Jessie Guidry, (firstname.lastname@example.org)