Biology Reference
In-Depth Information
9. Add 30
L of 10 mM dithiothreitol buffer to the gel and incu-
bate for 10 min at 60 °C.
10. Remove the dithiothreitol buffer and then leave the gel for
20 min.
11. Add 30
μ
L of 50 mM monoiodoacetic acid to the gel and incu-
bate 15 min.
12. Remove the monoiodoacetic acid.
13. Add 40
μ
L of 50 mM ammonium hydrogen carbonate buffer
and incubate for 15 min.
14. After removing the ammonium hydrogen carbonate buffer,
add 50
μ
L of 100 % acetonitrile and incubate for 15 min.
Repeat this step.
15. Volatilize acetonitrile using nitrogen gas.
16. Add trypsin solution to the gel and incubate for 2 h at 37 °C.
17. Add 10
μ
L of ultrapure water and then incubate for 2 h at
37 °C again.
18. Add 10
μ
L of 25 mM ammonium hydrogen carbonate buffer
and incubate for 10 min.
19. Add 20
μ
μ
L of 100 % acetonitrile and incubate for 10 min.
20. Add 20
μ
L of 10 % formic acid and incubate for 10 min.
21. Add 30
L of 100 % acetonitrile and incubate for 15 min.
22. Desalt the resultant sample solution using Zip-Tip and then
elute with 80 % acetonitrile.
23. Analyze the sample using matrix-assisted laser desorption
ionization time-of-fl ight mass spectrometry (MALDI
TOF-MS) using Voyager-DE STR.
24. Calibrate internal mass using trypsin autolysis products.
25. Identify the protein species by peptide mass fi ngerprinting
(PMF) on the nonredundant amino acid database of the National
Center for Biotechnology Information (NCBI-nr) using the
MASCOT search engine [ 54 ] ( see Notes 10 and 11 ).
μ
3.3 Notes
1. Stock 5 mL of 8 M urea with 2 % CHAPS solution in 15 mL
tube in −20 °C freezer. When used, add 14 mg of dithiothrei-
tol (Wako) to the solution, and then completely dissolve.
2. IPG buffer and IPG dry strip depend on the objective pI of 2DE.
3. Stock 10 mL of the buffer in 15 mL tube in −20 °C freezer.
4. For one trial of 2DE analysis, 2.5 mL of beer sample is needed.
Therefore, 50 mL of beer sample poured into the fl ask is enough.
5. Several proteins are specifi cally contained in the foam fraction.
Therefore, a complete degas is needed to analyze total beer
proteins
6. For more information, refer to the manufacturer's instructions.
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