Sample Preparations

When preparing samples for mass spectrometric analysis, it is best to limit sample manipulations. When dealing with solution samples, never bring the sample to complete dryness. Salts and detergents will interfere with MS analysis of solution samples. Based on extensive studies, we have found that SDS-PAGE or 2D IEF-SDS-PAGE is the best method for recovery of low quantities of protein.

Suggested method for staining gels with Coomassie Blue G-250

It is important to run and process the gel in as clean an environment as possible. Use thoroughly washed or disposable labware, wear gloves, and make sure reagents are fresh and uncontaminated.

You should clean a glass or plastic tray with detergent and rinse it thoroughly. You should wear clean, disposable gloves and never touch the gel bands directly. Never reuse stains and thoroughly acid wash staining trays if you reuse them.

  1. Remove the gel from the electrophoresis chamber and place into enough 0.5% Coomassie blue G-250 (made in 50% methanol/10% acetic acid) to cover the gel. We suggest using a new Petri dish or the top cover of a box of P-1000 pipette tips. Stain for about 5 minutes.
  2. Discard stain (note for protein chemistry work please do not reuse Coomassie solutions) and rinse briefly with high purity water to remove most of the residual stain in the tray.
  3. Destain with 40% methanol/10% acetic acid, replacing the solution every 10-20 minutes until faint bands are observed. Kimwipes rolled up into a ball can be added to speed up the destaining.
  4. Start destaining in high quality water (preferably Milli-Q water) until bands are very clean. Usually we destain overnight in water with several pieces of Kimwipes present. The gel will usually expand a little. Bands can now be excised and submitted for analysis.

Protein gels can also be stained with Gel Code Blue (Pierce). We have obtained nanogram-level detection of proteins with the Colloidal Blue Staining Kit (INVITROGEN). Please follow instructions from the manufacturer.

The recommended Silver Staining Protocol.

The recommended in-gel digestion protocol.

Examples

Below is an example of a SDS-PAGE gel stained with Coomassie G-250. Click on gel to see a larger picture of the gel.

gel.jpg (11028 bytes)

Generally, if a band is visible under these conditions, that is usually enough to obtain adequate MS data to perform a database search.