Proteomics Laboratory | Protocols

General recommendations for sample submission

The Lerner Research Institute Biomedical Mass Spectrometry Laboratory accepts samples from any institution. All investigators are strongly encouraged to discuss their project prior to sample submission with Dr. Belinda Willard (216-444-7170). Most proteins for internal sequence analysis will be submitted as bands run in polyacrylamide gels stained with Coomassie blue. Where sufficient material is available, the quality of the results may be improved by submitting a more abundant band rather than a less abundant. Care must be taken, however, to do good electrophoresis. In general, thin gels with well-focused bands give better yields and better results than thick gels with diffuse bands even though both contain the same amount of protein.

After staining and destaining, the investigator should photograph or scan the gel for their records and send a copy with the gel indicating the band or bands for analysis. We ask that the entire gel be submitted. This allows our laboratory to see the molecular weight markers, the resolution from other bands and the overall staining level. These observations greatly facilitate our ability to troubleshoot failed analyses.

The gel can be submitted wet, equilibrated into either water or 5% acetic acid in water. The wet gel can be brought to the lab in an appropriate container or placed in a heat sealable bag with all excess liquid poured off prior to sealing. Please include a completed sample submission form describing the sample in as much detail as possible, including the species that it was isolated from, approximate molecular weight, a brief description of how the protein was isolated, and a brief description of the purpose of the experiment (for example: identify a protein, obtain data for design of cloning experiments, confirm proper sequence of an expressed protein, etc.). Also, please give the sample a unique and informative name. We recommend the following format, initials-gel date-protein MW (ie BBW, 1-1-00 gel, p100). Sample names such as: "sample 1", "top band", "control", and "unknown" are not acceptable because of the confusion created by other submissions with the same name. Finally, include an activities code for billing.

Written reports are made for all analyses. Typical turnaround time, from submission of sample to preparation of report, is approximately three weeks. In general, samples are processed in the week they are received, and analyses begun the following week. If no peptides are observed in the digest then the investigator is notified at once. If peptides are observed then further analyses are carried out and data interpretation begins during the second week. Data interpretation and preparation of the report is generally completed during the third week. Bills are generated in the month that the report is written.

A recommended Coomassie blue staining procedure

Coomassie blue staining is a simple and reasonably sensitive method for visualizing protein bands in polyacrylamide gels. In our experiments, Coomassie staining can reliably detect approximately 0.5 pmol of BSA (0.05 ug) in a 0.75 mm thick mini-gel and our in-gel digestion procedure with capillary column LC-microelectrospray MS analysis can generate and sequence peptides from these samples. In 2D gel bands, we would estimate the limit-of-detection for Coomassie staining to be approximately 0.2 pmol depending on the sharpness of the band. This protocol uses a colloidal stain that is appears to be more sensitive than standard Coomassie stains.

In general, the only key step in the procedure is the method of fixing the gel. The gel must be fixed by a non-modifying, precipitation procedure such at the ethanol (or methanol)-acetic acid method used here. The amounts of solvents used, the times, the type of Coomassie blue are not generally critical. This procedure is based on the method used routinely in the Kinter lab. One may also note that handling of the gel should be minimized and that gloves should be worn at all times. One should also be careful with the tray in which the staining is carried out. It is possible to contaminate the gel with albumin, IgG, or milk if the tray is routinely used for Western blotting as wells. Attention to these details will minimize surface contamination of the gel.

Reagents

  1. Fixing solution: 50% ethanol, 10% acetic acid.
  2. Wash solution: MilliQ water
  3. Stain: Pierce Chemical Company's Gel code blue.
  4. Destain: MilliQ water.
  5. Storage solution: 5% acetic acid, 10% glycerol in water.

Procedure

As an overall note, the gels are stained in clean, glass trays. The trays are gently agitated during all steps to assure even treatment. All solutions are removed by aspiration to minimize handling of the gel during the procedure.

  1. After electrophoresis, fix the gel by soaking it in the fixing solution for at least 30 min.
  2. Aspirate off the fixing solution and wash the gel at least three times in water, at least 10 min per wash.
  3. Aspirate off the water and cover the gel with the Coomassie stain.
  4. Stain the gel at room temperature for 3 h to 24 h (we use overnight).
  5. Aspirate off the Coomassie stain.
  6. Destain by soaking the gel in water for approximately 1 h. Longer times may be necessary with gels stained overnight.
  7. Transfer the gel to a heat sealable bag.
  8. Scan the gel and add 3 - 5 mL of the storage solution.
  9. The gel can be stored for at least several months at room temperature.

An acceptable silver staining procedure

Silver staining is a more sensitive, but more difficult, method for visualizing protein bands in polyacrylamide gels. In our experiments, silver staining is anywhere from 10-fold to as much as 100-fold more sensitive than Coomassie staining. As a result, silver staining can be used to detect as little as 10 fmol of protein.

In general, the key limit on the use of silver stain procedures prior to in-gel digestion and mass spectrometric analysis is the type of fixing that is used. The commonly used reagent glutaraldehyde is an excellent fixer that is used in most of the high sensitivity procedure. Glutaraldehyde, however, acts by cross-linking the protein by modifying lysine residues and make a protein intractable for proteolytic digestion. Therefore, no matter what the details of the silvering and development steps, the gel must be fixed by a non-modifying, precipitation procedure such as the ethanol (or methanol)-acetic acid method described here. In many instances, this change will reduce the overall sensitivity of the protein detection.

One may also note that handling of the gel should be minimized and that gloves should be worn at all times. One should also be careful with the tray in which the staining is carried out. It is possible to contaminate the gel with albumin, IgG, or milk if the tray is routinely used for Western blotting as wells. Attention to these details will minimize surface contamination of the gel.

Reagents

  1. Fixer: 50% ethanol-10% acetic acid
  2. Reducer-sensitizer (Farmer's reagent, Electrophoresis 6:103-112, 1985): 1.5g potassium fericyanide + 3.0g sodium thiosulfate + 0.5g sodium carbonate in 1L water.
  3. Silver reagent: 0.5 g/L silver nitrate (AgNO 3 ).
  4. Developer: 3 g/L sodium carbonate + 200µL formalin per L. Add formalin just prior to use.
  5. Stop solution: 1% acetic acid.

Procedure

  1. Fix the gels in 50% ethanol-10% acetic acid for at least 30 min.
  2. Wash gels in water for 1 h with several changes of water.
  3. Sensitize gel with reducer-sensitizer for 5 min. Longer times are not recommended.
  4. Wash gels in water three times for 10 min each.
  5. Silver the gels in silver reagent for 30 min with shaking.
  6. Wash the silver reagent out of the dish and the gel for 2 min in a large amount of water.
  7. Develop:
    1. Cover the gel with the developer.
    2. Shake the tray and watch for the desired degree of development. Be ready to stop the reaction as the development reaches a desired point. The reaction does not stop immediately.
    3. Change the developer if it turns to a turbid brown color.
  8. Stop the reaction by rapidly removing the developer and adding the stop solution.
  9. Wash the gel in water.
  10. Reduce the gel with the sensitizer-reducer for 1 to 2 min as needed to remove any surface silvering or excessive background.
  11. If desired, the gel can be 'recycled' by retreating with the sensitizer-reducer for approximately 5 min and beginning the procedure at step 4 above.
  12. If the gel is done, be sure to rinse well with water to remove any residual sensitizer-reducer and prevent fading of the gel.
  13. Transfer the gel to a heat sealable bag.
  14. Scan the gel and add 3 - 5 mL of the storage solution.
  15. The gel can be stored for at least several months at room temperature.