A primary goal of working with gels intended for downstream LC-MS/MS analysis is to avoid contamination with foreign proteins. These proteins are typically human keratins derived from human skin or animal keratins from the clothes we wear. These proteins can be transferred to the gel or items which contact the gel (plates, buffers, stains, tools, etc.) via direct contact or by being exposed to the air, as dust in the air is composed of human skin cells.
Gels are not dangerous to work with, but it is beneficial to approach working with them as you would if you were working with materials which had to be kept sterile or were radioactive. That is, approach them with a mind-set aimed at minimizing contact and contamination.
Moden mass spectrometers are extremely sensitive and conventional workflows assay samples which often consist of less than one microgram of protein. Keratin contamination can easily overwhelm a sample, becoming the dominant protein component and possibly rendering the assay useless.
- We recommend the use of pre-cast gels from any of the major vendors. This is the first step to minimizing gel contamination with foreign proteins.
- Use the same care in preparing the electrophoresis apparatus and gel buffers as you would in excising spots or bands from the gel.
- Use nitrile gloves, do not use latex gloves.
- Wash your gloves frequently with water and shake dry or dry with a lint free wipe.
- Avoid direct contact with buffer reagents.
- Prepare buffers using clean glassware..
- Do not leave buffers exposed to the open air any longer than is necessary.
- Avoid working in a hood which pulls air into the hood, use a laminar flow or “dead air” hood instead.
- We recommend the use of Coomassie Brilliant Blue (Thermo LC6060) or MS-compatible silver staining (Thermo 24600) kits from a major vendor, this avoids potential contamination from user-prepared stains.
- If preparing the stain yourself, follow the same guidelines outlined above to avoid contamination.
- Transferring the gel from the plate to the staining dish is the most common source of contamination, try to avoid direct contact with the gel or with the solution in the staining dish.
- When staining, keep the dish covered to avoid exposure to air.
- Limit staining to the minimum time needed to visualize the band(s) of interest.
- Do not heat (e.g., in a microwave) to speed the staining process.
- Destain to reduce background.
Excising spots or bands
- Avoid direct contact with the gel.
- Many tools are available to assist with excision: scalpels work well for bands, Harris punches work well for spots, the Gel Company sells a variety of tools, including wire grid cutters to cut many bands from a gel lane simultaneously.
- Make sure the tools you work with are clean: rinse them regularly with water and dry with ethanol or a lint-free wipe.
- Rinse the tubes or plate wells into which the gel bands will be stored with water and dry with ethanol or acetonitrile.
- After placing the spot or band in the tube or well it is not necessary to treat them if they are to be shipped immediately.
- Gel bands can be shipped at ambient temperature.
If you plan to store the gel bands prior to shipping:
- destain with 50% ethanol in water, 50 C, 30 min; repeat twice more
- dehydrate with 100% acetonitrile, RT, 5 min, repeat twice more
- cover band with 100% acetonitrile and store at 4 C
- remove acetonitrile prior to shipping