Biology Reference
In-Depth Information
10. Leave the sample on ice for another 10 min in the dark.
11. At least two different sample amounts were used for assessment
of the labeling effi ciency in SDS PAGE. We routinely use 3 and
1 mg of protein. Add an equal volume of 2× SDS sample loading
buffer to the labeled protein and heat at 95°C for 5 min.
12. Run the sample on the 12.5% SDS-PAGE gel.
13. Scan the gel with the Typhoon imager.
14. Quantify the labeling of each sample using the ImageQuant
software.
15. Generate a volume report to check that labeling effi ciency is
similar across all the samples with the same dilution and load.
If labeling is comparable, proceed with the labeling of all sam-
ples for the fi rst-dimension IEF (see Note 10).
16. If the labeling is not comparable, poststaining of the gel using
a total protein stain such as silver or fl uorescent stain is carried
out to determine if it is due to differences in labeling effi ciency
or sample load inconsistency.
3.6. CyDye™ Labeling
for 2D DIGE
1.
Aliquot 50 mg of each sample into properly labeled microfuge
tubes. To prepare the internal standard, an equal amount of
all the samples in the experiment are mixed together to give a
pooled sample (see Table 1 ) (see Note 11).
2.
Incubate the samples with 1 mL of 400 pmol/mL CyDye™ on
ice in the dark for 30 min.
3.
Quench the reaction by adding 1 mL L -lysine with incubation
on ice in the dark for 10 min.
1.
Mix 50 mg of Cy3-labeled sample and 50 mg of Cy5-labeled
sample with 50 mg of Cy2-labeled internal standard to give a
total of 150 mg protein load/gel (see Table 1 ).
3.7. First-Dimension
Isoelectric Focusing
2.
Add rehydration buffer containing 7 M urea, 2 M thiourea, 4%
w/v CHAPS, 20 mM DTT, and 0.5% (v/v) pH 3-10 NL IPG
buffer, with trace amount of bromophenol blue, to a total
volume of 350 mL.
3.
The IPG strips are subjected to passive rehydration (remem-
ber—gel face down!) on the Immobiline DryStrip Reswelling
Tray for 12-16 h at room temperature in the dark.
4. Transfer the rehydrated strips to the Ettan IPGphor ceramic cup
loading strip holder (gel face up) for IEF on the IPGphor unit.
5.
Place electrode pads moisted with deionized water on each
end of the IPG strips for better contact between the gel and
electrodes.
6.
Cover the IPG strips with 4 mL of mineral oil.
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