Biology Reference
In-Depth Information
6. Once rehydration is fi nished, place paper strips moistened with
deionised water between the strip and the electrode using
clamps, towards the end corresponding to the anode (+).
7. Program the PROTEAN IEF cell system for the isoelectric focus-
ing according to the instructions of the system. For these samples,
a program at 20 °C with a fi nal voltage of 10,000 V (60,000 V-h)
was used. The maximum current per strip must be 50
8. Once the isoelectric focusing is fi nished, remove the strips
from the system and keep them vertically to dispose of most of
the mineral oil. Strips must be placed keeping the gel on the
top in the strip-holding system (rehydration/equilibration
tray) with the same order in which they were removed from
the system ( see Note 12 ).
9. At the time of performing the 2-DE, wash the strip twice
using the electrophoresis running buffer directly on the strip-
holding plate (rehydration/equilibration tray).
10. Equilibrate each strip with 5 mL of IPG equilibrium solution
containing 2 % (w/v) DTT for 15 min with gentle agitation.
Subsequently, perform the same procedure but using an equilib-
rium buffer with 2 % (w/v) iodoacetamide. Finally, wash with a
running buffer to remove the remnant equilibrium solution.
μ
A.
11. Prepare a 12 % resolving polyacrylamide gel ( see Note 14 ).
12. Insert the gel cassete assembly to the electrophoresis chamber
according to the manufacturer´s instructions and add enough
electrophoresis buffer to the top of the gel.
13. Place the strip previously equilibrated in steps 9 - 11 , on top of
the gel using clamps, avoiding the accumulation of bubbles
between the strip and the gel. This procedure must be done
preventing tearing of the strip and allowing the pH 3 end to
be located on the left end of the gel.
14. Gently moisten a piece of fi lter paper (10 × 5 mm) with 5
L of
weight marker (SDS-PAGE Broad Range markers Bio-Rad ® )
and place it on the left side of the strip.
15. Seal the system gently using the sealing solution and avoiding
the formation of bubbles. In order to do so, heat the solution
in a microwave oven and add 1-2 mL of it on the strip, previ-
ously positioned, until reaching the top of the glass. Use a
plastic Pasteur pipette.
16. Close the electrophoresis chamber and perform the electro-
phoresis procedure at 100 V.
17. Switch off the power supply and remove the gel carefully,
when the electrophoresis is fi nished.
18. Perform the staining procedure according to the previously
selected protocol. For carnation extracts, SYPRO Ruby-
Coomassie colloidal staining must be used (Subheading 3.3 ).
μ
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