Protein electrophoretic techniques

Because protein samples are actually ampholytes, when samples are loaded onto the gel and a current is appHed, the compounds migrate through the gel until they come to their isoelectric point where they reach a steady state. This technique measures an intrinsic physicochemical parameter of the protein, the pi, and therefore does not depend on the mode of sample appHcation. The highest sample load of any electrophoretic technique may be used, however, sample load affects the final position of a component band if the load is extremely high, ie, high enough to titrate the gradient ampholytes or distort the local electric field. 

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) An electrophoretic technique used for the separation of proteins. 

Separation of a mixture of proteins by electrophoretic techniques such as polyacrylamide gel, SDS polyacrylamide or iso-electric focusing usually results in a complex pattern of protein bands or zones. Interpretation of the results often involves a comparison of the patterns of test and reference mixtures and identification of an individual protein, even using immunoelectrophoresis (Figure 11.15), is very difficult. However, specific proteins can often be identified using an immunoblotting technique known as Western blotting. The prerequisite is the availability of an antibody, either polyclonal or monoclonal, against the test protein. 

After the initial separation by a conventional electrophoretic technique in a gel, the proteins are transferred (or blotted) electrophoretically from the gel to a membrane, usually nitrocellulose. The gel and the membrane, which has been previously soaked in a suitable electrophoretic buffer, are sandwiched between two electrodes. A voltage is applied, e.g. 100 V, and the proteins migrate from... 

Size-based analysis by CE provides similar information and comparable limits of detection to analysis by SDS-PAGE with Coomassie blue staining.120 129 The performance of both electrophoretic techniques for the analysis of polypeptides is far superior to size exclusion chromatography. Figure 9.7 shows the separation of SDS-complexed recombinant protein standards by CE. 

Much of the early work on the development of the proteins in tropical populations, utilizing mainly electrophoretic technique, have been summarized (E6). Using both the quantitative electrophoretic and qualitative immunoelectrophoretic methods to study the developmental patterns of... 

Thus, differences in functionality of these two suspensions cannot be related to protein quality as distinguished by the gel electrophoretic techniques used in this study. However, these data suggest that solubility of the major storage proteins, or their subunit components, contribute to foam capacity. In addition, other seed constituents, such as carbohydrate and ash (for example, field pea and pecan, respectively) may be equally involved (especially when the suspension pH is 1.5). 

Electrophoretic Methods. Little use has been made of electrophoretic techniques for the fractionation of the whey proteins. Column isoelectric focusing has been used to fractionate further the crude immunoglobulin fraction obtained by Smith s procedure (Josephson et al. 1972). Two major peaks, a shoulder, and two minor peaks were obtained, but no attempt was made to identify the components in the peaks. 

The electrophoretic techniques discussed up to this point are useful for analyzing proteins and small fragments of nucleic acids up to 350,000 daltons (500 bp) in molecular size however, the small pore sizes in the gel are not appropriate for analysis of large nucleic acid fragments or intact DNA molecules. The standard method used to characterize RNA and DNA in the range 200 to 50,000 base pairs (50 kilobases) is electrophoresis with agarose as the support medium. 

TMP Cattaneo, A Feroldi, PM Toppino, C Olieman. Sample preparation for selective and sensitive detection of soya proteins in dairy products with chromatographic and electrophoretic techniques. Neth Milk Dairy J 48 225 -234, 1994. 

In multiple myeloma, the neoplastic plasma cells secrete monoclonal proteins such as either IgG or IgA. Generally, either k or A. light chains are secreted. These M (monoclonal) proteins can be detected by serum and urine protein zone and immunofixation electrophoretic techniques, the latter providing better discrimination. The immunoglobulin levels exceed 25 g/L. Quantitation of immunoglobulins can be performed by rate nephelometry. 

The second main class of blood constituents used as genetic markers are the polymorphic enzymes. The enzymes of interest to the forensic serologist are primarily located within the red blood cell and are commonly referred to as isoenzymes. These can briefly be described as those enzymatically active proteins which catalyze the same biochemical reactions and occur in the same species but differ in certain of their physicochemical properties. (This description does not exclude the tissue isoenzymes that occur within the same organism however, our consideration deals only with those of the red blood cell in particular.) The occurrence of multi-molecular forms of the same enzyme (isoenzymes) has been known for several decades however, it was not until the Metropolitan Police Laboratory of Scotland Yard adapted electrophoretic techniques to dried blood analysis that these systems were catapulted to the prominence they presently receive (.2). For many of the forensic serologists in the United States, the use of electrophoresis and isoenzyme determination is a recently-inherited capability shared by only a few laboratories. 

It is now recognised that a substantial number of proteins, especially enzymes, are polymorphic in that they exist in the cell as multiple molecular forms differing in certain of their physico-chemical properties. Each form of a polymorphic enzyme is called an isoenzyme or isozyme. Electrophoretic techniques provide convenient methods whereby this protein heterogeneity can be investigated and the approach has been widely exploited to characterise parasites. In short, aqueous parasite extracts are electro-phoresed, or focused isoelectrically, and separated proteins are stained generally (usually with Coomassie Blue) or more specifically with a histochemical (enzyme) stain (the zymogram technique). Further details of individual procedures and the use of the approach in parasite identification are to be found in a number of recent reviews (104,258,413,536,615,856). 

Isoelectric focusing (IEF) is a steady-state zonal electrophoretic technique. Using IEF, proteins can be separated according to differences in their isoelectric points. The principles of IEF are described in Sections 8.10 and 8.11. 

The only other known study on the binding of A9-tetrahydrocannabinol to plasma proteins was performed with electrophoretic techniques on tritium-labeled material and human plasma (14). The compound was 90 -95% associated with lipoproteins. 

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