Table of Contents
Advertisement
7
Using Ettan DALTsix for electrophoresis
The unit should be placed close to a sink for easy rinsing and draining. The tubing leading to and from the heat
exchanger should be connected to a circulating water bath such as the MultiTemp III; the heat exchanger should
not be connected to a water tap or any other coolant supply that lacks pressure regulation. An EPS 601 Power
Supply should be placed conveniently near the electrophoresis unit.
WARNING! When using hazardous chemicals, take all suitable protective measures, such as wearing
protective glasses and gloves resistant to the chemicals used. Follow local regulations and instructions for
safe operation and maintenance of the system.
7.1
For a detailed description of the components of the SDS equilibration solution and the equilibration process,
please consult 2-D Electrophoresis: Using Immobilized pH Gradients (80-6429-60).
1
2
3
4
5
6
Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC
Preparing second dimension gels: equilibration and loading
Prepare SDS equilibration buffer. Just prior to use, add DTT to the buffer to a concentration of 1% (w/v).
Place the IPG strips in individual tubes with the support film toward the wall.
Add 10–15 ml of the DTT-containing solution to each tube. Typically, two 18 cm strips can be equilibrated
with 10 ml of buffer or two 24 cm strips can be equilibrated with 15 ml of buffer.
Incubate the strips for 10-15 min with gentle agitation. Do not over-equilibrate, as proteins can diffuse out
of the strip during this step.
Second equilibration. Prepare SDS equilibration buffer with iodoacetamide added to 2.5 % (w/v) and repeat
steps 3 and 4.
Before equilibration is completed, prepare the gel cassettes for loading by rinsing the top of the gel with
deionized water and draining. Before loading the IPG strips make sure that the gel surface and plates are dry.
Fig 7-1. IPG strip are loaded onto cassette and slid into place.
Using Ettan DALTsix for electrophoresis 7
7
Lay the prepared gel flat on a clean surface, short
glass plate side up.
8
Using forceps, remove the equilibrated IPG strip from
the equilibration solution and rinse with fresh SDS
electrophoresis buffer.
9
Holding one end of the IPG strip with forceps,
carefully draw it across the exposed top part of the
long gel plate until the strip is completely on the glass
plate and centered. Using a thin plastic spatula, ruler,
or spacer push against the plastic backing of the IPG
strip, not the gel itself, and slide the strip between the
two glass plates and down into contact with the
surface of the slab gel. The strip should just rest on the
surface of the gel. Avoid trapping air bubbles between
strip and the slab gel or piercing the second
dimension gel with the strip. By convention, the acidic,
or pointed, end of the IPG strip is on the left. The gel
face of the strip should not touch the opposite glass
plate. (See Fig 7-1).
21
Advertisement
Chapters
Table of Contents
