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- Run gel. If possible, use a precast gel. If precast gels are
not an option, filter acrylamide solutions prior to pouring the
gel and wash and dry glass plates in a dishwasher. Avoid using
Kimwipes. If wipes are needed, use low linting wipers such as
Kimtech Critical Task Wipers, which can be purchased from VWR
(cat. no. 21908-205). Run one lane with 0.5-1 ug BSA to be used
as a positive control. If doing an in-gel trypsin digestion for
the first time, I can provide a BSA positive control.
- Stain gel. To get the best mass spectrometry results, avoid
using silver staining. If choosing Coomassie Brilliant Blue as
your staining reagent, use Colloidal Coomassie G250 rather than
R250. A number of vendors now sell colloidal coomassie staining
solutions: BioRad (Bio-Safe), Pierce (GelCode Blue), Invitrogen
(SimplyBlue SafeStain).
- Pick spots/cut bands. Working in a dust-free environment and
wearing gloves and a lab coat, pick spots using a clean Pasteur
pipet/cut-off pipet tip, or cut out gel band using a clean razor
blade. Rinse cutting tool with dI H2O before touching
gel, and avoid using Kimwipes. If the gel is stained with Sypro
Ruby or Deep Purple, spots can be picked using a standard UV lamp.
Place gel plugs in clear polypropylene tubes/plates. If working
with gel bands, cut the band into 2 mm slices and combine into
one tube.
- Destain gel plugs/bands.
- Incubate 2.0 mm gel plugs with 100 ml
50 mM ammonium bicarbonate in 50 % methanol for 20 minutes
at room temperature. If working with gel bands, increase
volume relative to gel amount (I typically use 1 mL, and place
on a rocking platform.). If using Coomassie, increase destaining
time to remove as much stain as possible.
79 mg ammonium bicarbonate
10 mL methanol
10 mL H2O
- Remove methanol solution. Repeat. If using Coomassie,
repeat until methanol solution is no longer blue.
- Dehydrate/dry gel plugs/bands.
- Incubate 2.0 mm gel plugs with 100 ml
75 % acetonitrile for 20 minutes at room temperature. Again,
if working with gel bands, increase volume relative to gel
amount.
15 mL acetonitrile
5 mL H2O
- Remove acetonitrile solution. Dry gel plugs at
40 oC for 15-20 minutes. If working with gel bands,
increase drying time to ensure that gel is completely dehydrated
and “crisp”.
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