Biology Reference
In-Depth Information
we tried to use CA buffer as the extraction buffer composed of
calcium chloride in acetate buffer.
11. The 2-DE, image analysis of 2-D gels, and trypsin digestion of
2-D protein spots were performed essentially as described pre-
viously [
13
,
14
]. Briefl y, the colloidal CBB stained 2-D gels
were scanned (300 dpi, 16-bit gray scale pixel depth, TIFF fi le)
for image analysis using ImageMaster 2D Platinum imaging
software ver. 6.0 (GE Healthcare Bio-Sciences AB, Uppsala,
Sweden). The intensity of each spot was normalized as an aver-
age of the intensity of spots on the gel. Differentially expressed
(
p
< 0.05) protein spots present in three independent biologi-
cal samples were selected, digested with trypsin (Promega,
sequencing grade), and subjected to MS analysis.
12. MudPIT analysis were basically performed as described previ-
ously [
15
] using Partisphere strong cation exchanger (SCX;
Whatman, Clifton, NJ, USA) and Polaris C18-A. As peptides
were eluted from the microcapillary column, they were elec-
trosprayed into a LTQ linear ion trap mass spectrometer
(ThermoFisher, CA, USA) with the application of a 2.3-kV
spray voltage applied distally to the waste of the HPLC split.
Acknowledgments
This work was supported by National Research Foundation of Korea
Grant funded by the Korean Government (Ministry of Education,
Science and Technology) (NRF-2011-355-C00151) and Basic
Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education,
Science, and Technology (2011-0027211 and 2011-0018092).
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