Biology Reference
In-Depth Information
VARIOSKAN microtiter plate reader. Based on the standard
curve of BSA, determine the protein concentration of the
sample.
4. Stock the remaining desalted beer or wort sample in appropri-
ate tubes, such as a 15 mL tube, and freeze dry it.
5. Add 0.5 mL of dissolving buffer to freeze-dried samples, and
dissolve the sample completely using a vortex mixer.
1. Add an appropriate volume of sample solution containing
100
3.2.2 Two-Dimensional
Gel Electrophoresis (2DE)
and Staining
L of
0.1 % bromophenol blue solution to a 1.5 mL tube. Then, add
dissolving buffer to obtain a total volume of 300
μ
g protein (
see
Note 8
), 6
μ
L of IPG buffer and 10
μ
L, and mix
briefl y using a vortex mixer and then completely spin down.
2. Apply a total of 300
μ
L of the sample solution to an IPG dry
strip, and then rehydrate the IPG dry strip overnight.
3. Perform fi rst dimensional gel electrophoresis, isoelectric focus-
ing using a Multiphor II system. The electrophoresis condition
depends on the IPG dry strip used. For details, see the manu-
facturer's instructions.
4. Equilibrate the IPG strip using equilibration buffer containing
10 mg/mL dithiothreitol for 15 min, followed by equilibra-
tion buffer containing 25 mg/mL iodoacetamide (Wako).
5. Perform 2D SDS-PAGE using a precast XL 12-14 % gradient
gel using a Multiphor II system. For details, see the manufac-
turer's instructions.
6. Stain the gel with Silver Staining Kit, Protein. For mass spec-
trometry analysis, stain the gel with Silver Stain MS kit. For
detailed procedures, see the manufacturer's instructions.
μ
1. Excise the required protein spot on 2DE gel without
contamination (
see
Note 9
), and then stock the gel piece in a
1.5 mL tube.
2. Add 100
3.2.3 Mass-
Spectrometry Analysis and
Database Search
μ
L of decolorizing buffer to the gel and incubate for
5 min.
3. Remove decolorizing buffer, and then add 100
L of new decol-
orizing buffer to the gel. Repeat this procedure three times.
μ
4. Add 100
L of ultrapure water to the gel and incubate for 10 min.
5. Remove the ultrapure water, and then add 100
μ
L of new
ultrapure water to the gel. Repeat this procedure nine times.
6. Add 50
μ
L of 100 % acetonitrile and incubate for 15 min.
7. After removing the acetonitrile, add 50
μ
L of 50 mM ammo-
nium hydrogen carbonate buffer to the gel and then incubate
for 15 min.
8. After removing the ammonium hydrogen carbonate buffer,
add 100
μ
μ
L of 100 % acetonitrile.
