Biology Reference
In-Depth Information
2.5. In-Gel Digestion
1. 50 and 100% HPLC-grade acetonitrile (ACN). Store at room
temperature.
2. 5 mM ammonium bicarbonate (ABC) pH 8.0 in ddH 2 O. Store
at 4°C or prepare a 100 mM stock, aliquot, and store at −20°C.
3. 10 mM dithiothreitol (DTT) in 5 mM ABC. Prepare fresh.
4. 50 mM iodoacetamide (IAM) in 5 mM ABC. Prepare fresh.
5. 5% trifl uoroacetic acid (TFA) in 50% ACN. Prepare fresh.
6. 10 ng/mL sequencing-grade modifi ed trypsin (Promega) in
5 mM ABC pH 8.0. Prepare 500 ng-stocks in buffer provided
(50 mM acetic acid). Store at −20°C.
2.6. Matrix-Assisted
Laser Desorption/
Ionization Time-of-
Flight Mass
Spectrometry
1. Matrix solution: saturated aqueous 2,5-dihydroxybenzoic acid
(Bruker Daltonics).
2. Peptide external calibration standard (Bruker Daltonics).
3. Ultrafl ex matrix-assisted laser desorption/ionization time-of-
fl ight (MALDI-TOF)/TOF mass spectrometer (Bruker
Daltonics).
4. MTP AnchorChip target (Bruker Daltonics).
5. FlexControl software (Bruker Daltonics).
6. FlexAnalysis software (Bruker Daltonics).
1. Transfer-Blot tank (Bio-Rad).
2. Transfer buffer: 195 mM glycine, 25 mM Tris-HCl, pH 7.4,
20% (v/v) methanol. Prepare 10× transfer buffer in ddH 2 O
without methanol and store at 4°C. Dilute to a 1× solution
prior to transfer, adding 20% (v/v) methanol.
3. Tris-buffered saline with Tween-20 (TBS-T): 50 mM Tris-
HCl pH 8.0, 150 mM NaCl, and 0.1% Tween-20 (Sigma).
4. Polyvinylidene fl uoride membrane (PVDF) (Immobilon P,
Millipore, Bedford, MA), and 3-MM chromatography paper
(Whatman, Maidstone, UK).
5. Blocking buffer: 5% w/v low-fat milk in TBS-T.
6. Enhanced chemiluminescence (ECL) reagents (Perkin-Elmer
Life Sciences) and Fuji RX X-ray fi lm (Genetic Research
Instrumentation).
7. Primary antibody: appropriate primary antibodies chosen for
validation of MALDI-TOF results. Appropriate working
concentrations of the primary antibodies should be deter-
mined beforehand.
8. Secondary antibody: anti-mouse or anti-rabbit IgG-HRP
linked antibodies (GE Healthcare). Prepare freshly at a
dilution of 1:5,000 in TBS-T.
2.7. Validation
by Immunoblotting
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