Biology Reference
In-Depth Information
8.
Without disturbing the pellet, layer the coprecipitant onto
the pellet. The volume of coprecipitant used is 3-4 times
the volume of the pellet. Leave the tube on ice for 5 min.
9.
Carefully place the tube in the centrifuge as before and centri-
fuge at the maximum centrifugal force for 5 min.
10. Using a pipette, remove and discard the wash solution without
disturbing the pellet.
11. Next, add suffi cient volume of deionized water to cover the
pellet. Vortex to mix and ensure the pellet is dispersed well.
12. Following this, add 1 mL of the prechilled wash buffer and
5 mL of the wash additive. Vortex (hard) until the pellet is fully
dispersed.
13. Incubate the tubes at −20°C for 2 h. Vortex for 30 s once every
15 min during this step of incubation (see Note 6).
14. Centrifuge the tube at a maximum speed for 5 min.
15. Carefully remove and discard the supernatant. A white pellet
should now be visible. Allow the pellet to air-dry briefl y (for no
more than 5 min). Note that overdrying the pellet can result in
diffi culty in resuspension of the pellet.
16.
Finally, resuspend the pellet in 30 mL of 2DE rehydration
solution (see Subheading
2.5
) but without bromophenol blue.
If the resolubilization of the pellet is too slow (perhaps due
to a large pellet or a pellet being overdried), sonication can
help to speed up the resolubilization. Store the reconstituted
sample at −80°C.
3.4. Protein
Quantifi cation
Protein concentrations are determined using the Coomassie plus
protein assay kit based on the manufacturer's instructions with
some modifi cations. Perform the assay at least in duplicates (see
Note 7). Finally, measure the absorbance at 595 nm and determine
the protein concentration from a standard curve.
3.5. CyDye™-Labeling
Effi ciency Check Prior
to 2D DIGE
Always perform a preliminary 2D gel run for each sample. This will
facilitate the optimization of the separation parameters for the 2D
DIGE gel. It can also be used to check if there is any problem with
the sample. If the sample amount is limited, conduct a 1D PAGE
run to check that the calculated amount of protein to be used for
the 2D DIGE gel for a given pair of sample is similar based on the
staining intensity of the protein bands on the 1D gel. This is highly
recommended before proceeding with the CyDye™ labeling opti-
mization check as detailed in the next section.
For successful labeling, the pH of the sample needs to be in the
range of pH 8.0-9.0.
3.5.1. pH Adjustment
of Sample for CyDye™
Labeling
1.
Check the pH of the sample by spotting 0.1-0.5 mL on a pH
indicator strip (see Note 8).
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