Biology Reference
In-Depth Information
3 Protocol for Beer and Wort Proteomics Using Two-Dimensional Gel
Electrophoresis
Desalting and concentration of proteins are keys to obtaining
excellent gel images in 2DE of beer and wort proteins. To obtain
clear protein spots on the 2DE gel, appropriate desalting is espe-
cially important. The 2DE step is applied as usual. As an example,
the methods and protocol of sample preparation, 2DE, staining,
and mass spectrometry are shown below, although other methods
can also be applied. The strategy of beer and wort proteome analy-
sis and typical 2DE images of beer and wort are shown in Fig.
1
.
3.1 Materials
1. PD-10 column (GE Healthcare Biosciences, Tokyo, Japan).
2. Protein assay solution: Dilute (fi ve-fold) Bio-Rad Protein Assay
Kit II (Bio-Rad Laboratories, Tokyo, Japan) with ultrapure
water, and fi ltered it through fi lter paper.
3. Filter paper: Advantec, #2, 90 mm (Toyo Roshi Kaisha, Ltd,
Tokyo, Japan).
4. Bovine serum albumin (BSA): included in Bio-Rad Protein
Assay Kit II (Bio-Rad Laboratories).
5. 96-well plate: assay plate, 96-well, fl at bottom (well dia.
6.4 mm) (Iwaki, Tokyo, Japan).
6. VARIOSKAN microtiter plate reader (Thermo Electron
Corporation, Yokohama, Japan).
a
b
Mr.
(kDa)
Beer or wort
100
75
Decarbonation
50
37
25
20
Deionization by PD-10 column
(GE Healthcare Bioscience)
10
Lyophilization
C
Mr.
(kDa)
Resolution of lyophilized sample
66
45
31
Two-dimensional gel electrophoresis
22
14
Mass spectrometry analysis
Fig.
1
Strategy of beer and wort proteome analysis. (
a
) Experimental fl ow of beer and wort proteome analysis;
(
b
) Typical 2DE image of beer; (
c
) Typical 2DE image of wort
