Densities, various electrophoresis

TABLE 2 Electrophoresis of BSA adsorbed on glass at various densities and Ionic strengths... 

Electrophoretic Methods. Several electrophoretic procedures have been developed to fractionate or purify the various caseins (McKenzie 1971C Thompson 1971 Whitney 1977). Wake and Baldwin (1961) fractionated whole casein by zone electrophoresis on cellulose powder in 7 M urea and 0.02 ionic strength sodium phosphate buffer at pH 7 and 5°C. Payens and co-workers employed several somewhat different electrophoretic conditions for the fractionation and purification of the caseins on cellulose columns (Payens 1961 Schmidt and Payens 1963 Schmidt 1967). Three fractions, as-, k-, and /3-caseins, were separated at pH 7.5 and 30°C with 4.6 M urea-barbiturate buffer. The purification of asi-casein and the separation of the genetic variants of K-casein were accomplished by altering the electrophoretic conditions. Manson (1965) fractionated acid casein on a starch gel column stabilized by a density gradient at 25 °C. 

Fig. 12. Diagram of elution pattern of red cell acid phosphatase and various markers on Biogel P 60. The position of the various protein markers was determined both by optical density determination and by starch gel electrophoresis of the individual fractions (83). The experiment was carried out using a polyacrylamide gel (Biogel P 60, 50-150 mesh exclusion limit >60,000 Bio-Rad Laboratories, California) in 0.05 M tris buffer, pH 8.0, containing 0.08% (v/v) Tween 80 and 0.1% (v/v) 2-mercaptoethanol to stabilize the enzyme. Column 60 X 4 cm. Flow rate 20 ml/hr, 4 ml fractions. (A) OD at 280 nm, ( ) OD at 540 nm, ( ) LDH assay with p-nitrophenyl phosphate for AcP. From Hopkinson and Harris (85).

Quantitation may be done crudely on the spots separated by planar chromatographic techniques such as TLC or slab gel electrophoresis (see Chapter 13). One might compare the optical density, the fluoresence, or the degree of stationary phase fluoresence suppression by the unknown spot to a series of standards of known concentration. In contrast, the electrical signals from the variety of detectors used in various column chromatography instruments can be precisely, reproducibly, and linearly related to the amount of analyte passing through the detector cell. If all parameters of injection, separation, and detection are carefully controlled from run to run, and especially if appropriate quantitative internal standards are incorporated in the procedure, accuracy and precision better than +1 % may be attained. 

The properties of the crystalline enzyme from calf liver have been studied by Levine et al. (1969). Despite several recrystallizations the enzyme preparations showed up to three minor components on analytical ultracentrifugation, sucrose density gradient centrifugation, or polyacrylamide gel electrophoresis. This was not due to the presence of impurities, but rather to the occurence of multimers of the enzyme, i.e., monomer, dimer, trimer, and tetramer. Evidence to this effect was obtained from the close correspondence between protein content and enzyme activity in the fractions isolated by density gradient centrifugation and gel electrophoresis. Furthermore, the separated fractions slowly redistributed to yield analytical patterns similar to that of the native enzyme, indicating that interconversion between the various molecular species was taking place. 

Interaction of lipoproteins with various media and the electrical charge of the molecular complex are the main determining factors for separation of lipoproteins by electrophoresis. The significance of the protein moieties in such separation supplements ultracentrifugal methods and allows distinctions not possible with fractionation according to density alone. 

Addition of PMANa solution to a solution of DNA-polycation IPECs results in replacement of DNA by PMA polyanions, characterized by a higher linear charge density, and release of free DNA, which is detected by sedimentation as well as by electrophoresis data (not shown in Figs.). This substitution reaction is also typical for other IPEC s (see Sect. 2) a particular case is the separation of DNA or its fragments formed in the course of various chemical or biochemical treatments of IPEC(DNA/polycation). 

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