Protein zones, recovery from gels

Reverse stains In 1974, Wallace et al. developed reverse staining just to improve recovery of proteins for subsequent microchemical characterization. However, reverse stains are not suitable for quantitative analysis and staining of transfer membranes. The procedure is based on detection of proteins as transfer zones on a black background and used for visualization of proteins, passive elution of intact proteins from gels, and analysis by MS. Over the years, several different forms of procedures have been developed for reverse staining (Table 5.2). 

Elution systems. The recovery from IPG matrices would have to be electrophoretic for two reasons (a) an IPG gel would behave as a veak ion-exchanger (b) even extensively washed gels would still contain short, uncross-linked polyacrylamide-Immobiline chains which would be co-eluted with the protein if the latter v re to be extracted directly from an excised and ground gel zone. There are at least four elution modes, which are given belcw. 

A variety of rheological tests can be used to evaluate the nature and properties of different network structures in foods. The strength of bonds in a fat crystal network can be evaluated by stress relaxation and by the decrease in elastic recovery in creep tests as a function of loading time (deMan et al. 1985). Van Kleef et al. (1978) have reported on the determination of the number of crosslinks in a protein gel from its mechanical and swelling properties. Oakenfull (1984) used shear modulus measurements to estimate the size and thermodynamic stability of junction zones in noncovalently cross-linked gels. 

Following an electrophoretic run, the band from the tracking dye is often the only visible band. The detection of separated proteins and nucleic acids requires subsequent treatment of the separation pattern for visualization. This treatment may be performed directly on the gel, or may require a blotting step in which the entire separation pattern is transferred onto a thin membrane material. The choice of detection method depends on the concentrations of analytes in the separated zones and whether recovery of the purified sample is required. 

Figure 10. Recovery of proteins into a dialysis bag. Upon terminaticn of the IPG run, the gel strip containing the protein of interest is cut along the contours, chopped to pieces and loaded on top of a stacking gel in a preparative disc electrophoresis apparatias (here the glass tube has an inner diameter of 1.5 cm). After zone electrophoresis (usually 30-45 min at 4 C and 250 V), the protein is collected into the chamber having as a floor the dialysis membrane and as a ceiling the 5%T stacking gel, in a free liquid phase (20% sucrose in 100 mM Tris-acetate, pH 8.5) (Reproduced with permission from Ref. 48. copyright 1986, from Elsevier).

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