Biology Reference
In-Depth Information
10. When focusing is complete (see Note 11), stop the focusing
programme, remove the strips and drain the DryStrip cover
fl uid onto lint-free paper without delay.
11. If not proceeding to the second-dimension immediately, it is
possible to freeze the strips at −80°C (see Note 8).
Before the proteins can be separated according to their molecular
weight, it is fi rst necessary to break down the three-dimensional
structure of the proteins and to saturate the strip with sodium
dodecyl sulphate (SDS). This is done by equilibrating the strips
in a cocktail, which includes a pH buffer, urea, SDS and a dye for
monitoring the solvent front (see Subheading
2.2.5
). Any disul-
phide bonds are reduced with DTT and then alkylated with IA.
These equilibration steps are conveniently carried out in the rehy-
dration tray:
3.5. Second-
Dimension
Electrophoresis
1.
If the strips have been frozen, allow them to thaw to room
temperature.
2.
If using the rehydration tray for the equilibration steps, the
total amount of equilibration buffer needed will be 2 * [no. of
IPG strips] * 3 mL as there are two equilibration stages. This
is then split into two equal aliquots.
3.
Solid DTT is added to the fi rst aliquot at 0.5% (w/v), and solid
IA is added to the second aliquot at 4.5% (w/v). Both aliquots
are placed on the roller shaker to dissolve the added contents;
the IA-containing aliquot must be protected from light.
4.
Place the IPG strips gel-side up in the rehydration tray and
cover each strip with at least 2 mL of the equilibration buffer
containing DTT. Ensure all of the strips are completely covered
with the buffer.
5.
Cover the tray with the lid and place on the orbital shaker for
15 min.
6.
Remove the strips from the tray and immerse each one briefl y
in a measuring cylinder containing diluted run buffer (see
Subheading
2.2.5
), then drain onto lint-free paper.
7.
Clean out the rehydration tray and ensure it is completely clean
and dry.
8.
Place the IPG strips gel-side up in the rehydration tray and
cover each strip with at least 2 mL of the equilibration buffer
containing IA. Ensure all of the strips are completely covered
with the buffer.
9.
Repeat steps 5 and 6.
10. Add 750 mL of concentrated run buffer to the Ettan DALT
twelve
separation unit and add deionised water to the “7.5-L” mark.
Close the lid and turn on the pump to ensure thorough mixing
of the contents.
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