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Protocols for Prefractionation of Complex Protein Mixture for 2-D PAGE Using Reversed-Phase Liquid Chromatography

The utilization of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a widely accepted technique for the separation and analysis of proteins. Nevertheless, the presence of a composite mixture of proteins in a sample can result in a challenging scenario to obtain high-resolution separation. This issue can be tackled by using reversed-phase liquid chromatography (RPLC) as a pre-fractionation step, which diminishes the sample's complexity and enhances the quality of 2-D PAGE analysis.

RPLC is an intriguing method that relies on the differential solubility of proteins in a hydrophobic stationary phase. The separation of proteins based on their hydrophobicity is an imperative aspect, as it enables more hydrophobic proteins to be retained in the stationary phase for an extended duration than less hydrophobic proteins. Thus, it facilitates the segregation of a composite mixture of proteins grounded on their hydrophobic properties.

To execute RPLC, the sample is initially applied to the column, and the proteins are segregated based on their hydrophobicity. Then, fractions containing proteins are amassed and analyzed to determine protein content and purity. Subsequently, these fractions can be subjected to 2-D PAGE for further analysis.

The amalgamation of RPLC and 2-D PAGE is a potent strategy that yields enhanced resolution and sensitivity in the analysis of complex protein mixtures.

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.


  1. Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.
* For Research Use Only. Not for use in diagnostic procedures.
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