2D-GE Handling
First-time users of the BPF should contact the facility to ensure their samples are compatible with mass spectrometry.
2D-GE Handling
Contamination of samples with keratin proteins is a constant battle in mass spectrometry labs. To help minimize this problem, gloves and a lab coat should be worn at all times when working with gels and their associated reagents.
1. Virtually any 2D-GE gel should be compatible with down-stream protein identification. Pre-cast gels are suggested to help reduce keratin contamination. Pre-cast gels from BioRad are used routinely at the BPF.
2. Fresh staining reagents should be used (contaminants build up in re-used Coomassie stain).
Silver staining and Coomassie staining are both compatible with mass spectrometry-based protein identification.
Note: Silver staining kits should not contain gluteraldehyde as a fixing agent. Most commercial kits will state if they are MS friendly.
3. After staining, gels should be washed thoroughly in ddH20 prior to 2D-GE spots excision. Two washes at 15 minutes each should be sufficient. Longer washes are suggested for gels 1.5 mm and thicker.
4. 2D-GE spots of interest should be cut from the gel with a clean, sharp razor blade or spot cutter.
The 2D-GE spots should be cut directly on the edge of the staining region; no boarders of clear acrylamide should be left around the 2D-GE spot.
5. Cut 2D-GE spots should be placed in Eppendorf tubes (other brands of tubes sometimes have contaminants that interfere with the mass spectrometry). We suggest a new box of tubes be opened and individual tubes be carefully selected, again to cut down on keratin contamination. We discourage tubes being used from "community" containers.
6. The Eppendorf tubes can be stored at room temperature or 4 °C until they are prepared. Storing the 2D-GE spots at -20 °C or -80°C is discouraged as water in the gel will expand and cause the gel pieces to disintegrate, making the in-gel digestion procedure more difficult and reducing recovery of digested protein.
7. Label tubes clearly and place tape over writing
8. Tubes can be shipped via FedEx next day air.
Reagents to avoid
There are many common biochemical reagents that are not compatible with mass spectrometry or
HPLC and should not be present in the final sample. If the following reagents cannot be avoided during biochemical processing of the samples, the Proteomics Facility should be contacted to develop a strategy for removing the problematic reagents.
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Detergents: NP-40, SDS, Tween, Triton-X are not compatible with LC/ESI, MALDI, or HPLC.
DMSO at high concentration is not compatible with LC/ESI, MALDI, or HPLC.
Phosphate buffers are not compatible with MALDI
Glycerol at high concentration is not compatible with MALDI
Salts (i.e. NaCl, KCl) at high concentrations are not compatible with MALDI