Biology Reference
In-Depth Information
7. Dissolving buffer: 5 mL of 8 M urea (Wako, Japan) with 2 %
3-[(3-cholamidopropyl) dimethylammnonio] propane-sulfonic
acid (CHAPS) (Dojindo Laboratories, Kumamoto, Japan) and
0.28 % dithiothreitol (Wako, Tokyo, Japan) ( see Note 1 ).
8. IPG buffer (GE Healthcare Bioscience) ( see Note 2 ).
9. IPG dry strip (GE Healthcare Bioscience) ( see Note 2 ).
10. Multiphor II system (GE Healthcare Bioscience).
11. Equilibration buffer: 50 mM Tris-HCl, pH 8.8, 6 M urea, 30 %
v/v glycerol, 2 % sodium dodecyl sulfate (SDS) ( see Note 3 ).
12. Precast XL 12-14 % gradient gel (GE Healthcare Bioscience).
13. Silver Staining Kit, Protein (GE Healthcare Bioscience). Silver
Stain MS Kit (Wako).
14. Decolorizing buffer: 15 mM potassium ferricyanide and
50 mM sodium thiosulfate.
15. Zip-Tip (Nihon Millipore Ltd., Tokyo, Japan).
16. Voyager-DE STR (Applied Biosystems, Foster City, California
USA).
3.2 Methods
1. From a case of beer, open a beer sample packed in a bottle, can,
or any other packaging, and then pours it into a 300 mL coni-
cal fl ask ( see Note 4 ). Agitate the fl ask by hand several times
during foam formation to degas the beer. After no foam is
generated by shaking, leave the fl ask overnight in a wrap ( see
Note 5 ). In the case of wort, this procedure is not required
because wort is not carbonated.
2. Open the PD-10 column, and discard the buffer in the col-
umn. Load 5 mL of distilled deionized water into the column,
and run the water through the column. To achieve equilibra-
tion of the column, repeat the water loading four times, with a
total of 25 mL of the distilled deionized water. Then, load
2.5 mL of degassed beer or wort sample to the equilibrated
column. After running through the column, load 3.5 mL of
distilled deionized water in the column to elute desalted beer
or wort proteins ( see Note 6 ).
3. Determine the protein concentration of the desalted beer or
wort protein solution using the Bradford method with bovine
serum albumin as a standard [ 53 ]. For beer, no dilution is
required. For wort, dilute the sample three- or fi ve-fold with
distilled deionized water if required ( see Note 7 ). To deter-
mine the protein concentration, use 0.5 mL desalted samples.
Dilute standard BSA to 0.5, 0.4, 0.3, 0.2, 0.1, and 0.05 mg/
mL. Apply 10
3.2.1 Sample
Preparation
L of the diluted standard BSA and triplicate
samples to the 96-well plate. Then add 200
μ
L of protein assay
solution to each sample. After reacting for 10 min at room
temperature, measure the absorbance at 595 nm using a
μ
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