Biology Reference
In-Depth Information
DeCyder (left then right) and export the pick list coordinate
fi le (.txt) to the spot picker controller. Subsequently, open the
imported pick list and align the Ettan Spot Picker with the
reference markers according to the manufacturer's instructions.
Pick and collect spots in 96-well plates, drain the water, and
store at −20°C prior to MS analysis. Alternatively, excision of
spots from the post-stained gel can be done manually with a
gel-plug cutting pipette. The gel is best submerged under
1-2 mm of distilled water, and picking performed in a
dedicated clean area.
1. Ideally samples for MS analysis should be prepared in a clean
room or other clean area to avoid keratin and other
contamination.
2. Shake gel pieces in ddH
2
O for 15 min. Replace water with 50%
ACN and shake for a further 15 min. Repeat this step 3 times
until gel pieces are completely destained (see Note 12).
3. Remove the 50% ACN and dry in a speed vacuum for 15-20 min
(see Note 13).
4. Reduce the samples by adding suffi cient 10 mM DTT (in
5 mM ABC pH 8.0) to cover the gel pieces and incubating for
45 min at 50°C, with gentle shaking.
5. Remove the DTT solution and alkylate by adding enough
50 mM IAM (in 5 mM ABC pH 8.0) to cover the gel pieces
and incubating for 1 h at room temperature in the dark.
6. Remove the IAM solution and wash the gel pieces twice with
50% ACN for 15 min each.
7. Dry the gel pieces in a speed vacuum for approximately
15-20 min (see Note 13).
8. Digest the samples with trypsin. From a 500-ng/mL stock of
trypsin in buffer, dilute 100 times (to 5 ng/mL) to provide suf-
fi cient volume for all samples (10 mL of trypsin at 50 ng per
sample). Allow the trypsin solution to soak into the gel piece
and then add suffi cient 5 mM ABC pH 8.0 to cover the gel
piece. Place samples in an incubator or rocking heater block at
37°C and leave to digest overnight.
9. Briefl y spin the samples and collect the supernatant and transfer
to new siliconized tubes. Add suffi cient 50% ACN/5% TFA to
cover the gel pieces and agitate briefl y to aid peptide extrac-
tion. Remove the supernatant and pool together with the fi rst.
Repeat this step twice.
10. Speed vacuum to dryness. Samples can be stored at −20°C or
be directly analyzed by MS.
11. Resuspend peptides in 5 mL of 0.1% formic acid by gently
shaking prior to MALDI-TOF MS (or ESI MS/MS) analysis.
3.6. In-Gel Digestion
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