Biology Reference
In-Depth Information
In this example, where the 24 cm-IPG strips are rehydrated in
the presence of the protein sample, a maximum volume of 450 mL
is used:
1.
Pipette 40 mL of the Cy5-labelled sample into a 0.5 mL
Eppendorf tube. Add 40 mL of the Cy3-labelled sample and
40 mL of the Cy2-labelled internal standard. Repeat for all
samples as detailed in the gel plan.
2.
Add 22 mL of the 1.3 M-DTT solution to each of the sample
tubes and mix.
3. Modify the DeStreak solution by the addition of the IPG buffer—
added here at 2% (see Note 7)—to give a suffi cient volume for
all samples, i.e. for the ten gels prepared in this example more
than (450-22-(3 × 40)) mL × 10 = 3.08 mL is needed.
4.
Add 308 mL of modifi ed DeStreak solution to each tube, mix
the tube contents and stand on ice in the dark for 30 min.
5.
Level the clean and dry rehydration tray in a convenient posi-
tion on the bench and pipette each sample into an empty lane
and record which sample is in which lane.
6. Using tweezers, carefully peel off the backing strip for each IPG
strip, and place gel-side down on top of the sample. Ensure there
are no air bubbles trapped between the sample and the gel.
7.
If the strips are the correct way up, it should be possible to read
the serial numbers of the IPG strips. Record these on the Gel
Plan (see Table
1
).
8.
Cover each lane with approximately 2 mL of DryStrip cover
fl uid, then slide the lid into place.
9.
Protect from light and allow the strips to rehydrate overnight
(see Note 8).
3.4. Isoelectric
Focusing
This is the fi rst dimension of the separation, where voltage is
applied across the strips to separate the proteins according to their
pI. The voltage is increased in either a stepwise or gradient fash-
ion to fi rstly remove ionic material to the ends of the strips and
then to gradually move the proteins to their pI. The IEF pro-
gramme detailed in Table
2
was found to be suitable for use
with mouse livers. However, for any new study, the programme
must be optimised empirically as there are many factors that can
infl uence the separation, such as protein loading and concentration
of the IPG buffer.
IEF is conveniently carried out overnight. However, focusing
for too long can cause horizontal streaking in the gels. In the
programme detailed in Table
2
, the sole purpose of the last stage
(S6 in Table
2
) is to keep the proteins focused at low voltage until
it is convenient to remove them from the IPGphor. The programme
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