Two Dimensional Chromatography |
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Beckman Proteome PF2D system for 2-D chromatography The system is marketed as an alternative to 2-D gels. By combining separations based on charge and separations based on hydrophobicity, the system achieves resolution comparable to a 2D gel. IIf the same amount of different samples are loaded, they can be compared to see changes in expression of individual proteins. A fraction collector/injector unit allows the system to be largely automated after starting. Unlike a gel, this system collects intact proteins in solution for further analysis. The Beckman proteome PF2D system separates proteins by isoelectric point on a chromatofocusing column for the first dimension between pH 4 and 8.3. Proteins with pI above 8.5 pass through the column, and proteins with pI below 4 are eluted as one fraction at the end in 1M NaCl. The solvent used for the first steps are proprietary but is known to contain urea. Fractions covering 0.1 to 0.3 pH units are collected in a 96 well plate. This step takes about 3 hours and typically produces 30 fractions. The standard method collects fractions of 0.3 pH units. Then the collected fractions are separated by reverse phase in the second dimension, with elution by a gradient of acetonitrile containing trifluoroacetic acid; this takes 50 minutes for each fraction from the first dimension. The column is at 50°C for reproducibility and to help chromatography of hydrophobic proteins. Proteins eluted from the second dimension column can be collected. Up to 768 fractions can be collected in 96 well plates in one run. Detection in the first dimension is at 280 nm, where the buffers prevent the use of shorter wavelengths. In addition, the pH of the eluent from the column is measured. In the second dimension, detection is by absorbance at 214 nm. The manufacturer quotes a limit of detection of 500 pg, and a dynamic range of 104. For cell extracts, several milligrams of protein can be loaded, in a volume of up to 2 ml (repeated injections cannot be made for one sample). Recovery from both first and second steps is claimed to be 95%. The system is claimed to work well with proteins of sizes up to 150,000. A simple program gives a picture of peaks from the reverse phase column to produce an image with some similarities to a 2D gel, and highlights differences between samples. The program cannot normalize to correct for different sample loads or correct for different baselines in the second dimension run, so considerable manual interpretation may be needed. Samples need to be dissolved in or undergo buffer exchange into 1 ml of the starting buffer for the chromatofocusing column.
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