Biology Reference
In-Depth Information
11. With the second-dimension gel assembly in an upright
orientation add 1 mL of overlay agarose to the top of the gel
using a pasteur pipette (see Note 12).
12. Lay the second-dimension gel assembly horizontally on the
bench with the opening facing you and the smaller of the two
plates on top.
13. Using tweezers, place the IPG strip gel-side up on the inner
face of the larger of the two glass plates, with the “+” end facing
towards the left as you look at it.
14. Quickly stand the gel cassette upright with the smaller glass
plate in front (the “+” end of the IPG strip should now be
pointing to the right).
15. Carefully use the spatula to push the IPG strip down into the
agarose between the two glass plates (see Note 13).
16. Add more agarose if needed to completely seal the IPG strip
into place. Allow the agarose to cool and set.
17. Repeat this procedure for all of the other second-dimension
gels and IPG strips.
18. Introduce the gel cassettes into the separation unit (see Note 14).
19. Dilute a further 200 mL of the concentrated run buffer to
2 L with deionised water. Add this to the top of the unit, ensur-
ing that the fi nal liquid level is between the MIN and MAX
fl uid levels.
20. The Ettan DALT
twelve
can conveniently be programmed
to run overnight. It is advisable to focus the proteins at a
low constant power level (e.g. 0.5 W per gel) for the fi rst 2 h,
and then increase the wattage per gel to effect the separation.
Optimum conditions will be found empirically, but in the
work described in this chapter, a value of 2 W per gel was used
(see Note 15).
21. The dye front appears as a thin blue line travelling slowly down
the gel. When the dye front is close to the bottom of the gel
the Ettan DALT
twelve
should be stopped manually, and the
gels scanned without delay.
Gels must be scanned as soon as possible after the electrophoretic
separation. Any delay could lead to diffusion of the proteins away
from their tightly focused positions, which would broaden the
size of the spot and increase the chance of contamination with
other proteins.
Dust fl uoresces and scatters light, which can cause artefacts on
images and interfere with the subsequent quantitation. To minimise
the chance of this happening, gels should only be handled wearing
gloves, and these should be rinsed regularly with deionised water.
The scanner should be completely clean and dust-free.
3.6. Gel Scanning
and Image Analysis
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