Biomedical Engineering Reference
In-Depth Information
remainder of unused gel solution. This will set if left
undisturbed and sealed from the atmosphere, e.g. in a
capped tube.
10. Immediately before removing a 30
L aliquot of sam-
ple from the respective 1 mL fractions, pass each fraction
through a 26 gauge needle syringe twice. From experience
proteins in isolated raft fractions tend to aggregate. Passing
through a 26 gauge syringe needle gives a more homoge-
nous sample and so the 30
μ
L aliquot will be more repre-
sentative of the whole fraction.
11. Preparing a resolving gel at more than 20% acrylamide is
not recommended, as the stack can easily tear away from
the resolving gel when later removing the gel from the cas-
sette or excising the bands.
12. Do not boil samples solubilised in Tris/urea buffer. Pro-
teins in urea solutions heated above 37
◦
C can become
modified by carbamylation.
13. It is essential to load Laemmli buffered blanks of the same
volume of the samples on either side of all the samples.
This is to prevent band broadening and possible overlap of
adjacent samples during electrophoresis.
14. It is important to retain the stacking gel attached to the
resolving gel throughout the procedure. This is to ensure
the protein samples can be excised consistently and in their
entirety as a single band, at or near the interface between
the stack and resolving gels.
15. The time to stain is dependent on the sample concentra-
tion. For 10-30
μ
g protein samples, 30 min is usually suf-
ficient, although longer times may be required for less con-
centrated protein samples. Avoid staining overnight as the
dye can become difficult to remove from strongly stained
bands during the later in-gel digestion steps.
16. To excise the bands effectively, always use a new sharp
scalpel blade for each gel. Cut a line along the breadth of
the gel 1-2 mm above the bands in the 4% gel. Low-density
gels can be difficult to cut and some gentle sawing through
the cut may be required. Cut a similar line 1-2 mm below
the bands in the 20% gel. Excise each band by cutting ver-
tically 1-2 mm either side of each band. Use the tip of the
scalpel to collect the excised band and dice it into 1-2 mm
3
pieces. The gel band may become sticky if it begins to dry
out. To prevent this, periodically wet the scalpel and the
excised gel band with a droplet of water during cutting.
17. The trypsin will immediately become activated in neutral
or slightly alkaline solution. Use the solution as soon as
the aliquot has been diluted with TEAB. Do not stand the
solution for later use.
μ
Search WWH ::
Custom Search