Amersham Biosciences Hoefer DALT, User Manual, Page: 32 / 70

Loading and Running Second Dimension Gels Hoefer DALT System
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Amersham Biosciences
Iodoacetamide
alkylates sulfhydryl groups on proteins, preventing their re-oxidation
during electrophoresis. Protein re-oxidation during electrophoresis can result in
streaking and other artifacts. Iodoacetamide also alkylates residual DTT to prevent
point streaking and other silver staining artifacts. Iodoacetamide is introduced in an
optional second equilibration step.
N
OTE
The reaction between iodoacetamide and DTT creates hydroiodic acid.
When using iodoacetamide, the pH of the equilibration solution is 8.8 to
compensate for this acid production.
Tracking dye
(bromophenol blue) allows monitoring of electrophoresis.
Equilibration Steps
Use the gel number labels that were polymerized into the separating gel to help
orient the IPG strips. Conventionally, the acidic, or pointed, end of the IPG strip is
on the left, or label side.
N
OTE
To avoid protein contamination, wear gloves when handling the IPG strip
and hold only the protruding end of the gel backing film when moving the
strip.
1. Prepare SDS equilibration buffer (see page 43). Just prior to use, add DTT to the
buffer at a concentration of 100 mg DTT per 10 ml SDS equilibration buffer.
2. Place the IPG strips in individual tubes with the support film toward the wall. Screw
cap culture tubes (25
×
200 mm) work well.
3. Add 10 ml of the DTT-containing solution to each tube. Cap the tube, or seal it
with flexible paraffin film, and place is on its side on a rocker.
Note It takes
approximately 10 minutes
to prepare Agarose Sealing
Solution (page 43).
Prepare the solution during
IPG equilibration.
4. Equilibrate for 10 – 15 minutes. Do not over-equilibrate, as proteins can diffuse out
of the strip during this step.
An optional second 10-min equilibration with iodoacetamide will alkylate
sulfhydryl groups and prevent disulfide reformation. The second equilibration also
removes excess DTT which can lead to vertical point streaking when silver staining.
5. Second Equilibrium (optional) . Prepare a solution of 25 mg iodoacetamide per
10 ml SDS equilibration buffer. Add 10 ml of solution to each tube containing an
IPG strip. Cap the tube, or seal it with flexible paraffin film, and place is on its side
on a rocker to equilibrate, for 10 minutes.
I
MPORTANT
After equilibration, load IPG strips directly onto the DALT slab gels as
soon as possible.