2D DIGE Analysis of Serum After Fractionation by ProteoMiner Beads - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 193

the experiment. Therefore, the following are the recipe for
preparing the solutions:
Wash buffer (PBS buffer): 150 mM NaCl, 10 mM NaH 2 PO 4 ,
and pH 7.4.
Elution reagent: 8 M Urea, 2% (w/v) CHAPS.
Rehydration reagent: 5% (v/v) acetic acid.
2. DMF must be of high quality and anhydrous (specifi cation:
£0.005% H 2 O, ³99.8% pure). Once opened, DMF will start
to degrade, producing amine compounds. Amines will react
with the NHS ester fl uor and reduce the concentration of dye
available for protein labeling. Therefore, it is not recommended
to use DMF that is open for more than 3 months. Store the
opened DMF in a desiccator.
3. Removing the top cap prior to the removal of the bottom cap
before placing the column onto the capless tube is necessary to
relieve the pressure in the column. If the top cap is not removed
before the bottom cap, the solution in the column may leak
out immediately. This sequence in removal and replacing the
column cap need to be observed throughout the ProteoMiner™
fractionation step to avoid sample loss.
4. For plasma samples, there may be clumping after 1 h of binding.
Heparinized plasma is not compatible with ProteoMiner™.
5. Precipitate twice the amount of sample required for the DIGE
experiment as there will be sample loss in the process.
6. The samples can be incubated from 30 min to 1 week at −20°C
with minimal protein degradation or modifi cation.
7. The BSA standard used in the assay has to be diluted in the
same buffer as the sample being assayed. In this case, a diluted
rehydration buffer was used to ensure buffer components
are within the compatible concentration range as the protein
assay kit.
8. Cut the pH indicator into narrower strips so that less volume
of sample is consumed for pH testing.
9. Depending on the sample, usually an addition of 0.1-0.2 mL
of 1 M NaOH is suffi cient to adjust the pH of the sample to
pH 8.5.
10. It is recommended that CyDye™ labeling of samples is con-
ducted on the same day as testing the labeling effi ciency. In
our hands, we have experienced drifts in the pH of adjusted
samples after a day of storage.
11. Label slightly more protein for the Cy2 internal standard
than according to the intended number of gels (e.g., 10% more)
to allow for variation in pipetting. This is to ensure that there
is suffi cient Cy2-labeled protein for all the replicate gels.