Biology Reference
In-Depth Information
7. The reaction time for 50
g of protein and 400 pmol of CyDye
must be kept at 30 min or overlabeling will occur, and single
spot images may appear as double spots.
8. If it is not used immediately after IEF, each strip can be packed
to prevent exposure to air humidity and light and then stored
at −85°C until use.
9. To increase the solubility of TBP, 4% isopropanol is added into
the equilibrium solution, and the sample is sonicated for
30 min at room temperature. The reaction time must be kept
under 25 min. Handling of TBP should be performed in a
fume hood because it is very corrosive and fl ammable.
10. For fl uorescence scanning, the photomultiplier tube (PMT)
should be adjusted equally for the three CyDye emission wave-
lengths with respect to total spot volume intensity for all gels
with ImageQuant software. Expected PMT values are in the
range of 500-600 V. A PMT value over 600 V results from
undefi ned background signal. 2D DIGE gel plates on standby
for scanning should be kept in the dark at 4°C.
11. For image analysis, an adequate estimated spot number is 2,500
for plasma or serum because of the presence of high-abundance
proteins. If set over 2,500, the high-abundance spots will be
split into several areas, and then each area must be manually
merged. If set under 2,500, nearby spots might be assigned as
one area. In this case, the spot areas cannot be split by the
DeCyder program (v6.5.11). If the clinical sample is cells or
tissue, an adequate estimation of spot number is usually 3,000.
12. The threshold for the fold change is usually set to more than
±1.5-fold. In the case of 2D PAGE, the ratio cutoff is over ±2.0
due to gel-to-gel variation. In our results, a threshold of
±1.5 produced very large numbers of differentially expressed
protein spots.
13. The gel piece is easy to lose at this point because it becomes
smaller and transparent during the destaining procedure.
14. When the digest solution is not used immediately, store at
−70°C or lyophilize the solution to inhibit proteolysis.
μ
Acknowledgments
This study was supported by a grant from the National R&D
Program for Cancer Control (1120200 to YKP), National Project
for the Personalized Medicine, Ministry for Health and Welfare,
Republic of Korea (A111218-11-CP01 to YKP). The authors
declare no confl icts of interest. Corresponding author: Young-Ki
Paik (paikyk@yonsei.ac.kr).
Search WWH ::
Custom Search