The heterogeneity of phloem exudate proteins from different plants: A comparative survey of ten plants using polyacrylamide gel electrophoresis

Summary Proteins in sieve tube exudate from Ricinus communis L., Acer pseudoplatanus L., Aesculus hippocastanum L., Cucumis melo L., and two cultivars each of Cucumis sativus L., Cucurbita pepo L. and Cucurbita maxima Duchesne were fractionated and compared using polyacrylamide gel electrophoresis. Striking differences in major exudate proteins were displayed among the genera and species examined. Even cultivars within a single species, although showing general similarities, differed in some prominent proteins. Estimated molecular weights of the major exudate proteins from each plant are presented. The effects of reducing and chaotropic agents on the aggregation and subunit composition of exudate proteins from Cucumis sativus have been investigated. The problems involved in relating structure, function and biochemistry of P-protein are discussed.     

Fluorescent staining of sialidases in polyacrylamide gel electrophoresis and ultrathin-layer isoelectric focusing

Polyacrylamide gels were stained with the sialidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid showing the activity of Vibrio cholerae and Clostridium sordellii sialidases in the gels after electrophoresis. With this fluorogenic method minimum sialidase activities of 5 microU could be determined. The sensitivity of this staining is about 10,000-fold higher compared to protein-staining with Coomassie brilliant blue. For the visualization of other proteins than sialidases the specific sialidase staining could be followed by a protein-staining method in the same gel.     

Purification of NADPH-specific indole-3-acetaldehyde reductase from Cucumis sativus by two-dimensional native polyacrylamide gel electrophoresis

NADPH-specific indole-3-acetaldehyde (IAAId) reductase from cucumber ( Cucumis sativus L. 相似文献   

A new BF F variant by polyacrylamide gel isoelectric focusing

Summary Polyacrylamide gel isoelectric focusing followed by electroblotting with enzyme immunoassay was done for the investigation of allotypes of properdin factor B (BF) in serum from 326 Japanese subjects. A new BF F variant tentatively designated BF*Fb1 (b=basic) was detected, the isoelectric point of each band of homozygous BF Fb1 being higher than of BF FF. Family data were in accordance with transmission by mendelian inheritance. The allele frequencies calculated from 326 Japanese subjects were 0.7945, 0.1825, 0.0215, and 0.0015 for BF*S, BF*F, BF*Fb1, and BF*F075, respectively, with that of variant BF*Fb1 being a polymorphic frequency. The distribution of phenotypes fitted the Hardy-Weinberg equilibrium.     

Gc revisited: Six further Gc-phenotypes delineated by isoelectric focusing and by polyacrylamide gel electrophoresis

Summary Six newly observed Gc variants are described. The variants Gc 1A10, 1A11, 1A12, 1A13, and 1C11 have double band patterns. The anodal bands of these variants are susceptible to neuraminidase treatment. Gc 2A7 is a single band variant which is not altered by neuraminidase incubation. Polyacrylamide gel isoelectrofocusing with immunofixation and polyarcylamide gel electrophoresis appear to be efficient methods for the analysis of the Gc system.     

Simplified techniques for elution of proteins from polyacrylamide gel, staining, destaining, and isoelectric focusing

A simple and rapid electrophoretic method for the simultaneous elution of proteins in high yields from polyacrylamide gel slabs is described. The apparatus is simple and easily constructed. The method involves vertical elution from a horizontally placed gel across its thickness into buffer-soaked polyurethane foam. Even the slow-moving, high-molecular-weight protein ferritin is eluted in 1 h. The technique can also be used for rapid destaining as well as for simultaneous staining and destaining of the polyacrylamide gel which are completed in 15 and 30 min, respectively. Polyurethane foam strips have also been used to simplify the preparative isoelectric focusing technique and the subsequent elution of protein bands, obviating the need for Ultrodex, fractionating grid, sample applicator, and elution columns.     

Transferrin variants in sheep: separation and characterization by polyacrylamide gel electrophoresis and isoelectric focusing

Isoelectric focusing with carrier ampholytes in ultrathin polyacrylamide gels and polyacrylamide gel electrophoresis in a discontinuous buffer system were used for the separation of sheep transferrin variants. For identification of the different iron-binding sites of transferrin a stepwise urea gradient, different degrees of iron saturation and double one-dimensional electrophoresis were used. Isoelectric focusing results in an increased resolution of the Fe0-transferrin, Fe1-transferrin and Fe2-transferrin region. At the level of Fe0-transferrin and Fe1-transferrin the variants I, A, G, B, C, D, M, E, Q, P can be identified. The method is especially suitable for genetic studies. For screening purposes up to 108 samples can be separated within one run in an ultrathin gel.     

Transferrin variants in sheep: separation and characterization by polyacrylamide gel electrophoresis and isoelectric focusing*

Summary. Isoelectric focusing with carrier ampholytes in ultrathin polyacrylamide gels and polyacrylamide gel electrophoresis in a discontinuous buffer system were used for the separation of sheep transferrin variants. For identification of the different iron-binding sites of transferrin a stepwise urea gradient, different degrees of iron saturation and double one-dimensional electrophoresis were used. Isoelectric focusing results in an increased resolution of the Fe₀-transferrin, Fe₁-transferrin and Fe₂-transferrin region. At the level of Fe₀-transferrin and Fe₁-transferrin the variants I, A, G, B, C, D, M, E, Q, P can be identified. The method is especially suitable for genetic studies. For screening purposes up to 108 samples can be separated within one run in an ultrathin gel.     

Visualization of proteins after isoelectric focusing during two-dimensional gel electrophoresis.

A modification of P. H. O'Farrell's (1975, J. Biol. Chem.259, 4007–4021) two-dimensional gel electrophoresis is described. After isoelectric focusing, the cylindrical gels were fixed and stained with Coomassie brilliant blue R before sodium dodecyl sulfate-slab gel electrophoresis in the second dimension. The modification does not alter the protein patterns obtained, but provides sharper spots. In addition, bands are made visible before separation in the second dimension. Moreover, the modification helps to reduce the amount of ampholine in the dye front during electrophoresis.     

A new convenient column for gel electrophoresis,isoelectric focusing,and zonal electrophoresis

A simple preparative electrophoresis column that can be utilized for gel and zonal electrophoresis and isoelectric focusing has been constructed.     

Serial analysis of zein by isoelectric focusing and sodium dodecyl sulfate gel electrophoresis

Zein, the major storage protein of maize (Zea mays L.) endosperm, was extracted from a number of inbreds with alcohol plus a reducing agent. Isoelectric focusing (IEF) separated total zeins into 41 components, while sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separated total zeins into about 15 components. Each procedure gave characteristic patterns of zein bands for a number of maize inbreds. IEF and SDS-PAGE were used serially so that each band separated by IEF could be assayed as an individual SDS-PAGE sample. Some IEF bands revealed only a single band after SDS-PAGE, while others revealed two or more bands. A nomenclature system is presented which integrates the two separation systems with information about chromosome locations of zein genes, maize mutations which affect zein synthesis, and inbred sources for different zeins. SDS-PAGE of zein gives apparent molecular masses which vary widely according to the standards used and the properties of the gels, therefore an artificial nomenclature for identifying zein bands after SDS-PAGE is presented. The new nomenclature provides a flexible system which is useful and can be conveniently used in different laboratories.     

Analgesic and anti-inflammatory effects and safety profile of Cucurbita maxima and Cucumis sativus seeds

The premise of the pharmacology of natural product is to explore benefits of natural resources for the mankind. Medicines extracted from natural resources are considered as primary source for drug discovery. Thus, the current study was designed to evaluate the safety profile and explore the analgesic and anti-inflammatory activity of ethanol extract of Cucurbita maxima (C. maxima) and Cucumis sativus (C. sativus) seeds. These seeds are edible, good in taste and have been used for several therapeutic purposes. Acute toxicity of the seeds was evaluated by Lorke’s method while Eddy’s hot plate and tail immersion methods were used to assess analgesic activity in mice. Anti-inflammatory activity was evaluated by rat hind paw edema method. The seed extracts of C. maxima and C. sativus were found to be safe and showed significant analgesic and anti-inflammatory activity in comparison with the control group. The therapeutic effects of these extracts were almost comparable to aspirin and brufen. Therefore, the seeds can be used as effective analgesic and anti-inflammatory agents.     

Identification of macromolecules by quantitative polyacrylamide gel electrophoresis and isoelectric focusing: a comparison of three approaches.

Identification of macromolecules by zone electrophoresis has usually been based on differences in migration distances under a single set of electrophoretic conditions. Classically, it has taken the form of coelectrophoresis on gel slabs. In “quantitative” polyacrylamide gel electrophoresis (PAGE), separation conditions were standardized sufficiently to allow for identification of macromolecules between experiments on the basis of their relative electrophoretic mobilities, R_f ± σ_Rf. More reliably, molecular identity or distinguishability have been based on several R_f values at several gel concentrations (%T) and the linear relationship between log R_f and %T, the Ferguson plot. The slope (retardation coefficient K_R of this plot is desoriptive of molecular size while the γ-intercept (Y₀) is a measure of net charge. The joint 95% confidence envelopes for K_R and Y₀ may be used as criteria for identification of molecules. Distinction between two molecular species depends on the size and position of the two confidence envelopes or ellipses. By pooling estimates of residual varlance (scatter areund the regression line for the Ferguson plot) for several proteins, it is possible to reduce the size of the ellipses and improve the sensitivity of the method to distinguish elesely related species. The sensitivity of this method depends on the size and reprodueibility of the 95% confidence envelopes, and on the limitatiens in the number of electrophoretic fractionations that one is reasonably willing to invest. Any molecular identification problem therefore raises the implieit question whether to base distinction on migration distance, on R_f, or on the joint 95% confidence envelopes for K_R and Y₀ and related statistical (F test) eriteria. Further, in the event of inconsistent answers to the question of molecular distinguishability from the three approaches, we need rational criteria to select the “best” answer. These problems and some solutions are illustrated by the present study which was designed to determine whether the enzymatic digestion products of human growth hormone produced by subtilisin-B are or are not the same as those obtained by digestion with plasmin. It appears that the joint 95% confidence envelopes of K_R and Y₀ provide at this time the most discriminating criteria of distinction, indicating significant differences between nearly all the products of plasmin and subtilisin digestion of hGH. However, the lower resolution provided by the R_f criteria has the advantage that it allows one to group the products of the partial hydrolysis of hGH into “families” which may be associated with different ranges of specific bioactivities.