1. Sample Preparation
a) Harvest the HBL-100 cells by centrifugation at 2000g at 4°C for 10 min, wash twice in PBS and once in sample preparation buffer, spin down again to a "wet pellet," and determine the weight (W) of the pellet (mg). Freeze pellet at –80°C until use.
b) Lyse the cell pellets by addition of 0.841 × W mg urea and 0.304 × W thiourea, corresponding to 7 M urea and 2 M thiourea, respectively. Resulting volume (V) was assumed to be 2 × W.
c) Add 4% CHAPS and reduce the disulfide bonds by addition of 0.05 × V DTT (1.4 M stock solution, 70 mM final concentration)
d) Add 2.5% carrier ampholytes (Servalyte, pH 2.0–4.0) and additional protease inhibitors PMSF (1 mM final concentration) and pepstatin A (1.4 μM final concentration) and stir gently for 30 min at room temperature.
e) Clear the lysate by centrifugation at 200,000g for 20 min and freeze supernatant at –80°C until further use.
2. Reversed Phase High-Performance Liquid Chromatography
Liquid chromatography was performed with a Shimadzu LC-6 system using a Vydac C4 reversed phase column (150 × 2.1 mm, 5 μm, 300 Å) connected to a Vydac C4 guard column at a total flow rate of 0.5 mL/min (Note 2). Solvent A: 0.1% TFA in water; solvent B: 0.1% TFA in acetonitrile. The chromatograms were recorded at 280 nm.
a) Equilibrate the column with solvent A at a flow rate of 0.5 mL/min for 10 min.
b) Inject up to 1 mg of the sample (diluted 1:1 with solvent A) and wash the column carefully with solvent A for 20 min (stable baseline).
c) Start the step gradient that consisted of five steps with increasing concentrations of solvent B in solvent A. Step 1: 34%, 12 min; step 2: 38%, 10 min; step 3: 42%, 10 min; step 4: 46%, 10 min; step 5: 90%, 5 min.
d) Collect fractions automatically every 2 min.
e) Dry all fractions of each step in a SpeedVac concentrator.
f) Dissolve dried fractions in 20 μL SDS gel loading buffer for one-dimensional gel electrophoresis
g) Dissolve dried fractions in 2-DE buffer and pool fractions of each step for 2-D PAGE. The total volume of the pooled fractions should not exceed the loading capacity of a rod gel of the first dimension: 10–12 μL for an analytical gel and 40–50 μL for a preparative gel.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.