Biology Reference
In-Depth Information
g of protein per well and run the gels 1.5 cm. It
is important using gels of 1 mm thick (or less). Thicker gels
present problems for destaining and digestion. Leave one
blank well between samples to avoid contaminations. Fill these
wells with 1× Laemmli buffer.
7. Band destaining and cleaning is paramount for a good LC-MS
analysis. In these steps not only Coomassie, but also SDS and
other contaminants are removed. If there is still blue color
inside the gel pieces after three washings, continue with the
protocol, it will not interfere with protein digestion. This usually
happens when the gel pieces are not small enough.
8. Incubating the samples below 4 °C can stop trypsin digestion,
but better results are obtained when peptide extraction is done
just afterwards. We do not recommend spending more than a
couple of hours at this temperature or freezing the samples for
a later processing.
9. When using the 96-well plates in a vacuum manifold system a
special care should be taking regarding to the vacuum. The
vacuum should be the minimum to dry the membranes. The
vacuum should not be released sharply, because it can damage
the membranes and the plate. Bottom part of the wells should
not contact directly the waste tray at any moment. Collection
trays can hold up to 800
case load 90
μ
L before the fl ow-through reaches
the bottom of the wells, so waste tray should be emptied after
each equilibration/washing step.
10. In our experience the best results are obtained when the
desalted peptides are resuspended in a volume that is half of
the loaded micrograms. In our case we load 60
μ
g, which are
divided into two gel pieces containing approximately 30
μ
μ
g
each. Our setup injects 5
μ
L per run. So our recommended
L. If the amount of loaded pro-
teins or the HPLC injection volume varies, this resuspension
volume should change accordingly.
11. The specifi c gradient we use is the following:
resuspension volume is 15
μ
Time (min)
% A (0.1 % FA)
% B (90 % ACN, 0.1 % FA)
0
95
5
5
90
10
90
60
40
100
15
85
105
15
85
107
95
5
120
95
5
