Discontinuous electrophoresis

Electrophoresis Affinity electrophoresis (discontinuous system, 7% w/v total monomer concentration in the separation gel) was performed as previously described (6). 

Disc Electrophoresis. Resolution in zone electrophoresis depends critically on getting sample components to migrate in a focused band, thus some techniques ate employed to concentrate the sample as it migrates through the gel. The most common technique is referred to as discontinuous pH or disc electrophoresis. Disc electrophoresis employs a two-gel system, where the properties of the two gels are different. 

Another difference between other types of electrophoresis and disc electrophoresis is that the molecules in a sample do not start to significantly separate until entering the separating gel. A discontinuous gel system may be used with almost any type of 2one electrophoresis appHcation. 

Polyacrylamide gel electrophoresis is one of the most commonly used electrophoretic methods. AnalyMcal uses of this technique center around protein characterization, for example, purity, size, or molecular weight, and composition of a protein. Polyacrylamide gels can be used in both reduced and nonreduced systems as weU as in combination with discontinuous and ief systems (39). 

Assays. Protein concentrations were measured by the method of Bradford (18) and the various contractile protein ATPase activities by tRe method of Martin and Doty (19). Gel electrophoresis was carried out by the method of Ames (20) on 1.5 ran polyacrylamide slabs using the discontinuous SDS buffer system of Laemnli (21). Dried gels were scanned at 550 nm for densiometry measurements. 

The method of Kato and Nakai (27) for determining protein surface hydrophobicity was adapted for evaluating procyanidin binding to BSA and Gl. The procedure is based on the fact that the fluorescence quantum yield of cis-parinaric acid increases 40-fold when cis-parinaric acid enters a hydrophobic environment from a hydrophilic environment. The digestion of BSA by trypsin in the presence of procyanidin dimer, procyanidin trimer and black bean procyanidin polymer was evaluated by discontinuous sodium dodecyl sulfate (SDS) slab gel electrophoresis and a picryl sulfonic acid (TNBS) assay (28). 

The electrical current in an electrophoresis cell is carried largely by the ions supplied by buffer compounds. Proteins constitute only a small proportion of the current-carrying ions in an electrophoresis cell. Buffer systems for electrophoresis are classified as either continuous or discontinuous, depending on whether one or more buffers are used. They are further classified as native or denaturing, depending on whether their compositions maintain or destroy protein structure and activity. 

The choice of native electrophoresis system depends on the particular proteins of interest. There is no universal buffer system ideal for the electrophoresis of all native proteins. Both protein stability and resolution are important considerations in buffer selection. Recommended choices are the Omstein-Davis discontinuous system21-24 and McLellan s continuous buffers.25... 

It is important to bear in mind that an electrophoresis gel is an element in an electrical circuit and as such obeys the fundamental laws of electricity. Each gel has an intrinsic resistance, R, determined by the ionic strength of its buffer (R changes with time in discontinuous systems). When a voltage V is impressed across the gel, a current I flows through the gel and the external circuitry. Ohm s law relates these three quantities V = IR, where V is expressed in volts, I in amperes, and R in ohms. In addition, power P, in watts, is given by P = IV. The generation of Joule heat, H, is related to power by the mechanical equivalent of heat, 4.18 J/cal, so that H = (PI4.18) cal/sec. 

The Laemmli SDS-PAGE protocol is one of the most important analytical techniques in analytical protein separation. It is a system with discontinuous pH gradient (disc electrophoresis) and consists of a stacking and a separation gel different in acrylamide concentration and pH. The separation gel may be formed with homogenous acrylamide concentration or with an increasing gradient. 

SDS-polyacrylamide gel electrophoresis Protein samples were analyzed under denaturing conditions in a discontinuous gel system as described by Laemmli [13] using a 5% stacking gel and a 12% separating gel in a vertical gel system (BioRad, Mini Protean 11, CA, USA). 

Thus electrophoretic separation of proteins on PNIPAAm gel can be achieved, and the proteins can be recovered from the gel. However, the substantial technical difficulties we encountered made our vision of a simple swap of PNIPAAm gel for polyacrylamide gel in standard procedures impossible to realize. Thus, we discontinued further work on this project. However, the concept might still find use in cases of routine preparative gel electrophoresis where the problems and expense of solute recovery as described earlier were significant enough to warrant the developmental work required to make the PNIPAAm process practical. 

Protein concentrations were determined according to the method of Lowry et al. (30). Electrophoresis of proteins in polyacrylamide gels was carried out at 4°C, using the discontinuous buffer system No. 1 described by Maurer (31) and modified by Emert et al. (1). Protein was stained with 0.1% Coomassie Brilliant Blue R250 in a water-acetic acid-methanol (45 10 45) solution. Carbohydrates were stained with the periodic acid-Schiff (PAS) reagent using the method described by Lang (32). 

If the sample is applied as a spot, it splits up into a series of spots which lie farther apart as the time interval grows longer. This method with discontinuous application of the sample is called star electrophoresis because of the appearance of the stained substrate (P4). 

A last remark must be made about the use of the word continuous electrophoresis, covering collecting two-dimensional techniques in which both sample and background buffer feeding are continuous. As there is a two-dimensional technique with discontinuous application of the sample but a continuous feeding of buffer, the use of the term continuous is difficult, and again the terms collecting and star electrophoresis are preferred. 

A rapid two-dimensional separation of a microspot of protein solution under high field intensity is a new application of the two-dimensional principle. The various fractions emerge as a series of rays out of the application zone and hence the designation star electrophoresis which was introduced by us in connection with work on human serum proteins (P4). A similar discontinuous procedure was already mentioned by Strain in 1951 (S4, S5) for the separation of metal ions and recently used by Pucar (P8) and applied by Keler et al. (K1) to human serum. 

The most common classification scheme in electrophoresis focuses on the nature of electrolyte system. Using this scheme, electrophoretic modes are classified as continuous or discontinuous systems. Within these groupings the methods may be further divided on the basis of constancy of the electrolyte if the composition of the background electrolyte is constant as in capillary zone electrophoresis, the result is a kinetic process. If the composition of the electrolyte is not constant, as in isoelectric focusing, the result is a steady-state process. 

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