Preparing The Electrophoresis Unit - GE HEALTHCARE Ettan DALTsix User Manual

7 Using Ettan DALTsix for electrophoresis

7.2 Preparing the electrophoresis unit

10 Apply molecular weight marker proteins (optional). Apply the markers to a sample application piece in a
11 Seal the IPG strip in place. For each IPG strip, melt an aliquot of agarose sealing solution in a heating block
7.2
The Ettan DALTsix electrophoresis unit requires a total volume of about 5.0 l of electrophoresis buffer to fill both
the upper and lower chambers. For lab cast Laemmli gels, the lower chamber requires approximately 4.3 l of 1×
SDS electrophoresis buffer while the upper chamber requires 1.2 l of buffer with a higher concentration. Prepare
the required buffers as described below.
Electrophoresis buffer for Ettan DALTsix with lab-cast Laemmli gels
gel thickness
1.0 mm gels
1.5 mm gels
For the DALT Pre-cast Gels and Buffer Kit, only half of the 100× anode (lower) buffer is used for each run; the
slightly reduced buffer concentration should not affect the run conditions. To prepare the anode buffer, dilute half
the contents of the 100× anode buffer into 4.5 l of water. For the cathodic buffer, a single bottle of 10× cathode
(upper) buffer should be diluted to a final volume of 1.2 l with deionized water and the full amount added to the
upper chamber. Please consult the user instructions for the DALT Pre-cast Gels for detailed technical information
regarding the assembly of gels in their cassettes.
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volume of 15-20 μl then cover the piece with 50 μl of agarose sealing solution. Pick up the application piece
with forceps and place next to one end of the IPG strip. The markers should contain 0.2-1.0 μg of each
component for Coomassie blue staining and about 10-50 ng of each component for silver staining.
or boiling water bath. (Tip: an ideal time to carry out this step is during IPG strip equilibration). Allow the
agarose to cool slightly and slowly pipette the solution across the length of the IPG strip taking care not to
introduce or trap bubbles. It will flow down between the glass plate and the support film and seal the IPG
strip in place (see Fig 7-2). Agarose should also be used to seal any gap between the side of the gel and a
side spacer. Allow a minimum of 1 min for the agarose to cool and solidify.
Fig 7-2. Adding agarose overlay.
Preparing the electrophoresis unit
anodic buffer
(lower chamber)
1×SDS
electrophoresis buffer
1×SDS
electrophoresis buffer
cathodic buffer
vol (l) (upper chamber)
4.3 2× SDS
electrophoresis buffer
4.3 3× SDS
electrophoresis buffer
Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC
vol (l)
0.8
0.8