Preparing Protein Extracts for Quantitative Two Dimensional Gel Comparison

This unit provides methods for protein extraction and removal of interfering compounds for subsequent analysis of the proteins by two-dimensional (2-D) gel electrophoresis (unit 10.4). An important caveat is that these methods are intended only for two-dimensional separations in which the first dimension is isoelectric focusing (IEF). Thus, it is essential that the sample preparation process address the chemical constraints inherent in the IEF process and be reproducible between extractions to allow quantitative analysis of the resulting 2-D gels, the latter being crucial for quantitative proteomics analysis using 2-D electrophoresis (e.g., units 10.4 & 22.2). The procedures described in this unit address the first consideration by solubilizing the proteins in a solution similar to that used during IEF (i.e., a strongly denaturing solution of low ionic strength). The extraction solution usually contains high concentrations of urea and sometimes ancillary chaotropes such as thiourea, as well as nonionic and zwitterionic detergents, a disulfide bond reducer, and other reagents such as carrier ampholytes (used here as a buffer).

While simple dilution of pure protein samples in such solution is convenient as a sample preparation process, the situation is much more complicated with complex samples where the concentration of many interfering compounds (namely anything other than proteins) must not exceed a critical threshold. This threshold will depend upon many parameters (e.g., amount of sample loaded, geometry of the 2-D electrophoresis setup) and is therefore very difficult to predict. Some cellular components (e.g., DNA) strongly interfere with isoelectric focusing and must always be removed unless present in very low amounts (e.g., in mitochondrial preparations). Other components (e.g., salts, lipids, neutral polysaccharides) interfere much less severely and need to be removed only when highly abundant in the biological material of interest. Consequently, the optimized extraction process is strongly sample-dependent and cannot be described for every type. Nevertheless, the protocols in this unit serve as general guidelines and provide good starting points for sample preparation optimization.

Methods are listed according to type of biological material, starting with animal cells, nuclei, and bacteria (see Basic Protocol 1), animal tissues (see Alternate Protocol 1), plasma or serum (see Alternate Protocol 2), and other biological fluids (see Alternate Protocol 3). These are followed by extraction techniques for plant tissues and cells (see Basic Protocol 2), and finally subcellular organelles other than nuclei (see Basic Protocol 3).

NOTE: Refer to protocols concerning electrophoresis on immobilized pH gradient (IPG) gels and second dimension electrophoresis of IPG gels for more information on 2-D gel electrophoresis (unit 10.4).

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