Tips for SDS-PAGE Gel Handling
Contact: Phil Gafken
Contact phone: (206) 667-1051
Training Format: How-to
First-time users of the resource should contact the facility to ensure their samples are compatible with mass spectrometry.
SDS-PAGE Gel Handling Tips
- 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.
- Virtually any SDS-PAGE gel should be compatible with down-stream protein identification. Pre-cast gels are suggested to help reduce keratin contamination. Pre-cast gels from Invitrogen and BioRad have been used routinely at the Hutch.
- 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.
- After staining, gels should be washed thoroughly in ddH20 prior to band excision. Two washes at 15 minutes each should be sufficient. Longer washes are suggested for gels 1.5 mm and thicker.
- Bands of interest should be cut from the gel with a clean, sharp razor blade. The band should be cut directly on the edge of the staining region; no boarders of clear acrylamide should be left around the band.
- Cut bands should be place 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.
- The Eppendorf tubes can be stored at room temperature or 4 °C until they are prepared. Storing the bands at -20 °C or -80 °C is discouraged as water in the gel bands will expand and cause the gel pieces to disintegrate, making the in-gel digestion procedure more difficult and reducing recovery of digested protein.
- A blank region of the gel, approximately the size of the gel band of interest, should be submitted with the sample(s). This will act as a blank control.
- Label the tubes well! (initials, date and sample name)
- 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.
- 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