Biology Reference
In-Depth Information
2.
Picking lists may be generated with the corresponding gel
analysis software. In our laboratory, we use DeCyder 2D 7.0
(GE Healthcare) or Melanie 2D Gel Analysis Software (
http://
us.expasy.org/melanie/
).
3.
Before punching out gel spots for MS analysis, it is advisable to
make holes as coordinate markers along the sides of the wet
gel. Rescanning the gels at the 1:1 scale before and after spot
picking shows that the excision of gel pieces is done correctly.
The gels are fi rmly attached to the low-fl uorescence glass
support to prevent them from sliding during scanning and for
better spot picking (
1, 7
).
4.
Acceptable conditions for destaining gel pieces vary widely,
depending on the stain and the staining method, ranging from,
for example, (a) 25 mM ABC, 50% ACN at 37°C for 30 min
with shaking (
8
), (b) two washes with ACN/25 mM ABC
(70/30) (
9
), (c) 200 mM ABC, 40% ACN, destaining twice at
37°C for 30 min (
10
), and (d) 200 mM ABC, 50% ACN at
37°C for 45 min (
11
). Using high concentrations of ABC might
require addition of larger amounts of TFA in the acidifi cation
solution to drop the pH after the digestion step, thus producing
higher salt amounts that might impact microcrystal formation
on a MALDI target if a prior desalting step is not included in
the procedure.
5. For small gel pieces and low molecular weight proteins, it might
be important to minimize predigestion time during which a
gel plug is covered in solution. A study using metabolically
radiolabeled recombinant proteins showed that peptide recovery
from 0.5 mm gels was low and variable during fixing and
staining (
11
). Hence, it might be important to keep gels and
gel plugs for longer-term storage in a dry form (
9
). A multichan-
nel automatic pipette can be used for a higher sample through-
put. In order to reduce the chance of cross-contamination
between sample wells, it is convenient to slice the plate-covering
matt parallel to the sample columns or rows and open the matt
portion over one plate lane at a time when the solvent exchanges
are performed with a multichannel pipette.
6.
At this point, gel plugs should be small, hard, and white. Quick
sample evaporation in a vacuum evaporator will assure that gel
drying is complete. Thin gel-loading pipette tips or Hamilton
syringes are convenient tools for solvent removal while keeping
small colorless gel plugs in place. For dehydration of small
robotically picked gel plugs, it is better to use a smaller amount
of ACN (approximately 50
L) and then remove it entirely in
a vacuum evaporator. Alternatively, solvent handling and evap-
oration can be performed using a Hamilton syringe through
the pinholes made in a sample plate matt or a tube cap to prevent
μ
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