Protein Identification from SDS-PAGE Gel Bands
Protein samples can be subjected to either one- or two-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Typically we recommend loading 0.5-2 ug of protein, but more may be required for complex protein mixtures. After staining, the bands (1D SDS-PAGE) or spots (2D SDS-PAGE) are excised, alkylated and then proteolytically digested using an enzyme of your choice. The resulting peptides are then analyzed by HPLC/MS/MS.
The bands highlighted in green represent 500 ng to 1 ug of purified BSA and provide an example of “good” band intensities. Fainter bands do usually yield identifications but often at lower confidence.
Tips When Processing and Handling Gels for Proteolytic Digestion
The gel may be stained with coomassie brilliant blue R-250, colloidal coomassie blue G-250 or with a mass spectrometry compatible silver stain (such as Invitrogen SilverQuest, #LC6070 or Sigma Proteosilver kit, #PROTSIL1-1KT). Please consider the following guidelines when processing and handling your gel:
- Gels with 1 mm thickness work best.
- Wear gloves at all times and change them often during gel handling.
- Handle the gel as little as possible and keep it covered at all times.
- Avoid touching the gel with anything that has not been carefully cleaned.
- DO NOT use staining trays that have been used for Western blotting technique. Blocking agents (BSA or skim milk, etc) are not easy to remove and often suppresses the identification of other proteins.
- Only transfer the gel onto surfaces (light box, glass plate, etc.) that have been carefully cleaned.
- Once the band/spot of interest has been located, using a clean scalpel blade, cut the gel as close to the stain as possible. Cutting the gel while it is in the destaining solution minimizes contamination.
- Use a clean pair of forceps to transfer the gel slice into a 1.5 ml microcentrifuge tube. There is no need to include any buffer/water or other solvent in the tube.
- Gel pieces may be stored for a few days at 4°C until submission for analysis. Alternatively, they can be stored for months at -80°C until ready for analysis.
Neglecting these guidelines will almost always result in the identification of keratin. Please include an image of your gel when submitting. If you are submitting the entire gel, please provide a map of the gel indicating which bands you would like excised.
Note for 2-D gels: When using alkaline conditions (pH>8), sample must be alkylated prior to isoelectric focusing.
A cut and digested gel band will be analyzed with our hybrid quadrupole time of flight mass spectrometer coupled to a UPLC. A "bottom-up" approach is used in which the peptides characteristic of a protein are identified.
Post translational modifications on the protein of interest can also be studied. If this kind of study is desired, please consult with Dr. Virginie Sjoelund or Lmbcr_help@ouhsc.edu before submitting your sample.
Any problems with iLab read the help manuals below: