Minor Determinants, pages

M— DETERMINANTS AND MATRICES. 1. The Formation and Expansion of Determinants, pages 377-380. 2. Minor Determinants, pages 380-383. 3. Differentiation of a Determinant, pages 383-384. 4. General... 

For many years, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods have been used as an essential tool to determine the hydrodynamic size, monitor product purity, detect minor product or process-related impurities, and confirm batch-to-batch consistency of protein and antibody products. ITowever, gel-based techniques have several limitations, such as lack of automation, varying reproducibility, and a limited linear range. SDS-PAGE is also labor-intensive and generates large volume of toxic waste. Most importantly, the technique does not provide quantitative results for purity and impurity determination of proteins and antibodies. 

Fig. 2. NAPI facilitates H2A, H2B release from nucleosomes that are on positively coiled DNA (A) but not negatively coiled DNA (B). The positively coiled DNA (6.0 kb) with a superhelical density of + 0.05 and negatively coiled DNA (6.0 kb) with a superhelical density of -0.05 were reconstituted with lysine, arginine-labeled histones H3, H4, H2A, H2B by NaCl dialysis from 2.0 M to 1.2 M to 0.6 M to 0.1 M NaCl over a 14 h period. The samples were incubated with NAPI at 35 °C for 5 min and applied to a 5-20% sucrose/100 mM NaCl/40 mM Tris, pH 7.8 gradient. After sedimentation at 200,000 X g for 5 h, fractions were collected and the distribution of DNA (bottom panel) was determined on agarose gel and the distribution of protein (top panel) on SDS-PAGE followed by fluorography. These data are unpublished observations (V. Levchenko and V. Jackson). The deg-H2A is degraded H2A in which a 15 amino acid peptide of the C terminal has been proteolytically removed. When H2A, H2B is no longer present in a nucleosome, the C terminal region is sensitive to proteolysis [126] from a protease which is a minor contaminate in the NAPI preparation.

The best method to determine the success of antibody production to the minor components was made by two dimensional SDS-PAGE and immunoblotting (24). A comparison of the antisera (day 112 antisera) from the three groups demonstrated that the cascade immunization antisera detected a number of minor components (Figure 4C, arrows) which were not observed with the conventional or passive antisera (Figure 4B and D). It was clear from these results that the cascade antisera was far superior in its spectrum of antibody reactivity and, in fact, was comparable or superior in detection of ECPs to silver stain (Figure 4A). Although silver stain appeared to have an improved detection of certain low MW or basic proteins,... 

The other major class of proteins of the grape berry are the Thaumatin-like proteins (TLP) (Tattersall et al., 1997). In addition to the main TLP isoform called VVTL1, another minor TLP form can be detected (Pocock et al., 2000). These proteins show, respectively, an apparent MW of 24 and 25 kDa by SDS-PAGE, but their MW, determined by electrospray ionization-mass spectrometry, (ESI-MS) is 21.272 and 21.260kDa (Pocock et al., 2000). Moreover, the MW of the TLP identified by the proteomic analysis of the whole Cabernet sauvignon... 

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