Protein ID Solutions Sample Prep Guide
53
In-Gel Tryptic Digestion with Agilent Kit
6
Figure 7
Protein In-Gel Tryptic Digestion Kit procedure
Add 30 µL
alkylation buffer
1D Gel
Add 200 µL
destaining solution
Add 30 µL
reducing buffer
Add 200 µL
destaining solution
1. Place excised protein
band in receiving tube.
2. Destain gel slice.
(2 × 30 min at 37°C)*
3. Reduce.
(10 min at 60°C)*
4. Alkylate.
Perform in the dark.
(1 hr at room temp.)*
5. Wash.
(2 × 15 min at 37°C)*
Steps 3-5 are optional, but recommended
Add 50 µL
100% ACN
Add 10 µL activated trypsin or enough to cover gel
Swell 15 min
Add digestion buffer (25 µL for spot or 50 µL for band)
6. Shrink and dry gel
slice. (25 min)*
7. Digest.
(4 hr at 37°C or overnight at
30°C)**
Excise protein band
Notes:
* Remove and discard
solution afterward
(peptides are in gel)
** Retain solution
afterward
(peptides are in
solution)
Summary :
Protein ID Solutions Sample Prep Guide 53 In-Gel Tryptic Digestion with Agilent Kit 6 Figure 7 Protein In-Gel Tryptic Digestion Kit procedure Add 30 µL alkylation buffer 1D Gel Add 200 µL destaining solution Add 30 µL reducing buffer Add 200 µL destaining solution 1.
Tags :
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