Biology Reference
In-Depth Information
2.4 In Gel Digestion
of Protein Spots
1. Quick-CBB plus (Wako Pure Chemical Industries, Ltd., Osaka,
Japan) or Silver Stain Kit for Mass Spectrometry (Thermo
Fisher Scientifi c).
2. 1 M Ammonium bicarbonate: Weigh 67.9 mg and add 1 mL
of ultrapure water. Prepare before every use.
3. Dehydration buffer: 50 % acetonitrile, 25 mM ammonium
bicarbonate. Mix 1 mL of acetonitrile, 50
μ
L of 1 M ammo-
L of ultrapure water.
4. Trypsin Gold (Promega): Dissolve in 100
nium bicarbonate, and 950
μ
μ
L of ultrapure
water. Divide aliquots and store at −80 °C.
5. Protease Max (Promega): Divide 10
μ
L into each tube and
store at −80 °C. Before use, add 990
μ
L of 25 mM ammonium
bicarbonate to make a 0.1 % solution.
2.5 Mass
Spectrometry and
Protein Identifi cation
1. Zip-Tip C18 (Millipore Corp., MA, USA).
2.
α
-CHCA, Sigma-Aldrich
Corp., MO, USA): Add 50 % acetonitrile/0.1 % trifl uoroacetic
acid (TFA) to prepare a saturated solution (
see
Note 2
). Dilute
saturated
-Cyano-4-hydroxycinnamic acid (
α
-CHCA to 1:4 with 50 % acetonitrile/0.1 % TFA
just before use.
3. Opti-TOF 384 well Insert (123 mm × 81 mm) (Life
Technologies Corp., CA, USA).
4. 4800 MALDI TOF/TOF Analyzer (Life Technologies).
5. Mascot software (Matrix Science Inc., MA, USA).
α
3
Methods
3.1 Protein Sample
Preparation
1. Extract allergens from the plant specimens. Homogenize the
plant specimens and add an adequate volume of extraction
buffer. Rotate for more than 3 h to overnight at 4 °C and cen-
trifuge at 8,000 ×
g
for 10 min. Filter the supernatants using a
0.45
m fi lter.
2. Measure the protein concentration of the extracts with a Pierce
BCA Assay Kit or 2-D Quant Kit, in accordance with the man-
ufacturer's protocols. Divide into aliquots and store at −80 °C.
3. Purify and desalt an amount of extract containing 50
μ
μ
g pro-
teins with a 2-D Clean-Up Kit (
see
Note 3
).
4. Dissolve the protein pellet by vortexing in a 500
L of DeStreak
Rehydration Solution to obtain a protein concentration of
0.1
μ
μ
g/
μ
L. Divide the protein solutions into 25
μ
g aliquots
and store at −80 °C
