Combining Chymotrypsin/Trypsin Digestion to Identify Hydrophobic Proteins from Oil Bodies - Plant Proteomics: Methods and Protocols - page 179

2.3 Solutions
for In-Gel Digestion

Prepare fresh solutions for each in-gel digestion.

1. Washing buffer: 0.1 M ammonium bicarbonate. Weigh
119 mg of ammonium bicarbonate into a small glass beaker
and dissolve in 15 mL of water.
2. Washing buffer/acetonitrile (ACN): 0.1 M ammonium bicar-
bonate/ACN (1:1) (v/v). Mix equal volumes of washing buf-
fer and ACN in a glass test tube. For in-gel digestion of 20
samples, 10 mL of washing buffer/ACN is needed ( see Note 8 ).
3. ACN ( see Note 8 ).
4. 50 mM ammonium bicarbonate. Dilute the washing buffer
twice. For in-gel digestion of 20 samples, 4 mL of 50 mM
ammonium bicarbonate is needed. To be cold enough for fur-
ther use, keep the solution at 4 °C.
5. DTT solution: 10 mM DTT, 0.1 M ammonium bicarbonate.
Weigh 4.8 mg of DTT into a test tube and add 3 mL of wash-
ing buffer.
6. Iodoacetamide (IAA) solution: 55 mM IAA, 0.1 M ammo-
nium bicarbonate. Weigh 30 mg of IAA into a test tube and
add 3 mL of washing buffer. Keep the solution in a dark place.
7. Trypsin solution: Trypsin 12.5 mg/L in 50 mM ammonium
bicarbonate. Dilute the stock solution of trypsin (1 g/L) 80
times with cold (4 °C) 50 mM ammonium bicarbonate to
reach the fi nal concentration of trypsin 12.5 mg/L ( see Note 9 ).
For one sample, according to the amount of gel pieces,
30-60 L of trypsin solution is needed. Prepare the solution

immediately before use. Keep it at 4 °C.
8. Chymotrypsin solution: Chymotrypsin 12.5 mg/L in 50 mM
ammonium bicarbonate. Prepare the solution of chymotrypsin
as in previous step ( see Note 9 ). Keep the solution at 4 °C.
9. Protease mixture solution: Trypsin 6.25 mg/L, chymotrypsin
6.25 mg/L in 50 mM ammonium bicarbonate. Mix equal
volumes of trypsin and chymotrypsin solutions in 50 mM
ammonium bicarbonate. Keep the solution at 4 °C.
10. Extraction solution 1: 35 % (v/v) ACN, 5 % (v/v) formic acid
(FA). In a glass test tube, mix 3.0 mL of water, 1.75 mL of
ACN ( see Note 8 ), and 250

μ

L of FA.
11. Extraction solution 2: 70 % (v/v) ACN, 5 % (v/v) FA. In a
glass test tube, mix 1.25

μ

μ L of water, 3.50 mL of ACN

L of 5 % FA.
12. ZipTip pipette tips with C18 reversed-phase. We purchase
ZipTip pipette tips from Millipore.
13. ZipTip wash solution: 50 % ACN in water. Mix 2 mL of water
and 2 mL of ACN in a glass test tube ( see Note 8 ). Prepare
immediately before use and sonicate in a water bath for 5 min
to remove air bubbles.

( see Note 8 ), and 250 μ