Determination of molecular weights and Stokes' radii of non‐denatured proteins by polyacrylamide gradient gel electrophoresis. 2. Determination of the size of stable and labile molecular weight variants of enzymes from plant sources

Abstract

weight variants of enzymes from plant sourcesUnder certain conditions in polyacrylamide gradient gel electrophoresis (PAGGE), a linear correlation between the logarithm of the size of calibration proteins (log MW or log RJ and the square root of their migration distance (m) can be observed; slope and intercept of the calibration curve depend on the duration of electrophoresis; linearity, however, is maintained over a wide range (4-60 h, 200 V) (Rothe and Purkhanbaba, Electrophoresis 1982,3,33-42.) Using this method the reaction of plant isozyme systems penetrating a linear polyacrylamide (PAA) gradient gel was investigated: lactate dehydrogenase (LDH) from potato tubers behaves similarly to animal calibration proteins. The enzyme exhibits 3 subbands with molecular weights of 144 000, 150 000 and 156 000 (+ 1.7 %), respectively. The enzyme system "shikimate oxidoreductase" (SORase) behaves significantly different: during PAGGE it splits into 2 major bands (-60 000 and -1 10 000, respectively) consisting of several subbands. The results of our experiments show that SORase is present in vivo as a complex system. We believe that this complex is partly indentical with the so-called pre-chorismate complex.

2-D native-PAGE/SDS-PAGE visualization of an oligomer's subunits: Application to the analysis of IgG

“…The 2-D native-PAGE/SDS-PAGE method can be carried out with separation of proteins in the firstdimensional gel by alkaline native-PAGE [12][13][14], neutral native-PAGE [15], acidic native-PAGE [3], or native-PAGE at a wide range of other pHs [16][17][18]. Furthermore, for analysis of an oligomer of unknown molecular weight, the separation of protein mixtures in gradient gels of polyacrylamide [19][20][21][22][23][24][25][26][27][28][29] or agarose-acrylamide composite gels [30,31] in the first-dimensional native-PAGE will facilitate the estimation of the molecular weight of the native macromolecular assembly before denaturation and separation of the subunits in the second-dimensional SDS polyacrylamide gel. Alternatively, Ferguson analysis [32] will also facilitate the estimation of molecular weights of native oligomers in acrylamide or agarose gels [33][34][35].…”

Ionic liquids as buffer additives in ionic liquid-polyacrylamide gel electrophoresis separation of mixtures of low and high molecular weight proteins

“…The progress of proteomics is strongly dependent on the development of protein separation techniques and MS technology. [10][11][12][13] The ordinary gel-based electrophoresis techniques, SDS-PAGE 14-17 and 2-DE, 15,16,18 Blue-native 19,20 and Native-PAGE 21,22 are important and the most commonly used methods for protein separation. 2 A number of methods have been used for protein separations, which includes liquid chromatography, [3][4][5] size exclusion chromatography, 6 capillary electrophoresis [7][8][9] and gel electrophoresis.…”

Concentration gradient used in double-stranded DNA separation by capillary electrophoresis

“…Concentration gradient, which has been successfully used in SDS-PAGE for purification and separation of proteins by slab or rod electrophoresis [22,23], is a reasonable alternative to enhance the performance of separation medium on DNA analysis. Existing theories can validate this approach [9].…”