Biology Reference
In-Depth Information
experimental designs is represented by the number of gels
which is higher in a two-color experiment.
11. Just prior to use, add to the rehydration stock solution the
appropriate amount of IPG buffer (0.5% v/v), 65 mM DTT, a
trace of bromophenol blue, and the samples. The volume of
the rehydration solution depends on the IPG strips' length
(i.e., for 24-cm IPG strips, the rehydration solution volume
required per strip is 450 mL). Make sure the IPG buffer matches
the pH gradient of the strip used for IEF.
12. Select the strip holders corresponding to the IPG strip length.
Wash each holder with detergent to remove residual protein
and rinse with distilled water. Before use, the holders must be
completely dry.
13. The IPG cover fl uid volume to apply on the strip depends on
the length of the holder used. Add the oil until the entire IPG
strip is covered.
14. It is possible to rehydrate strips in the absence of protein sam-
ples. In this case, protein samples are loaded after rehydration,
using a cup loading technique.
15. A typical IEF protocol generally proceeds through a series of
voltage steps that begins at a relatively low value. Voltage is
gradually increased to the fi nal desired focusing voltage, which
is held for up to several hours. A low initial voltage minimizes
sample aggregation. It is important to remember that focusing
parameters for different pH gradients and different protein
loading need to be optimized.
16. Single percentage gels offer better resolution for a particular
MW window. When a gradient gel is used, the separation inter-
val is wider and spots are sharper because the decreasing pore
size functions to minimize diffusion. Remember that stacking
gels are not necessary for 2D gels.
17. Take care not to introduce bubbles and do not allow the aga-
rose to cool or solidify before placing the strip.
18. By convention, the acidic end of the strip is placed on the left.
19. Recommended running condition:
16 h overnight run, 15°C (2 W per gel).
-
-
8 h duration, 15°C (4 W per gel).
-
4 h duration, 15°C (8 W per gel).
-
The run is fi nished when the bromophenol blue dye front
reaches the bottom of the gel.
20. The excitation and emission wavelength used for all three
CyDyes are listed in Table 1 .
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