Quantitative measurement of protease activities in slab polyacrylamide gel electrophoretograms

A simple, rapid, and inexpensive method for the quantitative measurement of protease activity in slab polyacrylamide gel electrophoretograms is described. An electrophoretogram containing separated test samples and dilutions of a standard protease was placed in contact with a thin (0.7-mm) agar gel slab containing gelatin substrate. After 30 min incubation at 37°C, the unhydrolyzed gelatin was precipitated with saturated ammonium sulfate for 10 min. The developed substrate slab proteolytic zymogram was placed between two clear plastic sheets for analysis by densitometry at 525 nm. A standard graph of peak height in the densitometric scan against quantity of standard protease in micrograms or in proteolytic units was plotted. When measurements of a test sample were made in six independent experiments from the initial linear part of the standard curves the mean ± standard deviation was 3.2 ± 0.2 proteolytic units.     

Photoduplication and fluorescence measurement in polyacrylamide gel electrophoresis.

A simple method of photoduplication of gel electrophoresis, visualized with fluorescent reagents, is described. The procedure is convenient and rapid and requires no camera or expensive equipment. Using electrophoresis duplicating paper (Kodak), positive prints suitable for documentation or publication may be obtained. With usual photographic paper, negative prints may be obtained, allowing reliable measurement by scanning. The technique may be applied to protein or nucleic acid electrophoresis.     

A sensitive polyacrylamide disc gel method for detection of proteinases

To enable direct detection of proteinase activities subsequent to electrophoresis, a technique utilizing the incorporation or diffusion of protein substrates into polyacrylamide disc gels was developed. Denatured insoluble substrates, casein or hemoglobin, were added to acrylamide solutions prior to polymerization of the gel mixture. Alternatively, soluble protein substrates were diffused into gels after electrophoresis. In either case, an incubation period ensued at the pH optimum of the proteinases to allow for their detection. Classification of resolved proteinases was accomplished subsequent to electrophoresis by incubation of gels in media containing either synthetic substrates, as the naphthylamide derivatives, or specific inhibitors of the enzymes. Separation of purified trypsin from chymotrypsin, and proteinases in preparations of seminal plasma and mouse blastocysts homogenates demonstrated the efficacy of the method at the submicrogram enzyme level.     

Assay of plant proteins with bicinchoninic acid for high resolution two-dimensional polyacrylamide gel electrophoresis

A method is described for estimating proteins in the same plant tissue sample that is solubilized for separation by two-dimensional polyacrylamide gel electrophoresis. The method uses a modified bicinchoninic acid (BCA) protein assay procedure and a modified standard urea solubilization buffer to estimate microgram values of unknown protein concentration, in the presence of 9 M urea and 4% Nonidet P-40, from a linear standard curve. A method for a quantitative determination of protein concentration by BCA in a sample containing 9 M urea and 4% Nonidet P-40 is also described. This method is effective for the determination of proteins in minute non-green and green plant tissue, and is especially designed for vegetative and floral shoot apices, and the primordia of inflorescences.     

Direct detection of rutin-degrading isozymes with polyacrylamide gel electrophoresis

Rutin-degrading enzymes (RDEs) specifically hydrolyze the glycosidic linkages of rutin, producing quercetin and rutinose. Here we report a reliable and sensitive polyacrylamide gel electrophoresis and staining method for the detection of RDE isozymes, which is based on the aqueous solubility difference between rutin and quercetin, as well as the ultraviolet absorbance of quercetin. With this novel method, we achieved a detection limit of 12 ng with 107 U of RDE activity, enabling us to detect at least five RDE isozymes in tartary buckwheat seeds.     

A simple method for detection of neurosecretory products after polyacrylamide gel electrophoresis

Neurophysins, a family of cystine-rich proteins found in the hypothalamic-neurohypophysial system can be located histologically with aldehydefuchsin. A method is described whereby the aldehyde-fuchsin technique has been used to locate neurophysins after polyacrylamide gel electrophoresis.     

A simple electronic apparatus for the analysis of radioactively labeled gel electrophoretograms

Radioactively labeled gel electrophoretograms are generally analyzed either by slicing the gel and dissolving the individual pieces in a fluor for scintillation counting or by exposure to photographic emulsions. In this report, we describe a technique for fast quantitative analysis of such electrophoretograms, based on counting individually β-or γ-particles emerging from the gel surface and locating their position to ±0.6 mm. The apparatus which employs a position-sensitive single-wire gas proportional counter is simple to construct and operate with readily available electronics.     

Elsie 4: quantitative computer analysis of sets of two-dimensional gel electrophoretograms

We have developed and refined a system for quantitative computer analysis of two-dimensional polyacrylamide gel electrophoretograms. The system, named Elsie 4, is based on one described by Vo et al. (Anal. Biochem. 112, 258 (1981]. It is highly automated. Elsie 4 can find, and measure the intensity of, almost any spot resolvable on two-dimensional gels, including spots visible only as shoulders off larger spots and spots so close together that there is no valley between them. It can automatically match the spot patterns of different gels, potentially without the need for a user to provide landmark matches. The matches between paired gels let us follow the synthesis of any spot through a set of gels. Information about a group of matched spots can be obtained by referring to any spot in the group. There is generally no need for a standard or reference gel. Data for two experiments can be combined and compared by matching any gel in one experiment with any gel in the other. There are ways to automatically find possible mismatches in sets of gels. Scans and the results of the analysis can be shown on an image displayer. The programs use function libraries; this helps ensure consistency and increase portability. The programs and functions can be linked together in many ways; this lets users build custom programs for analysis of specific experiments.     

Improvements in polyacrylamide gel slicing

An improved polyacrylamide gel slicer has been devised that provides rapid uniform slicing with a precision of 4–6%. The advantages of this type of slicer are: The gel is sliced directly from the electrophoresis tube; gel diameter and length can vary with no modification of the system; and gels with a range of acrylamide concentrations can be fractionated with no pretreatment of the gel.     

Staining for protease activity on polyacrylamide gels

A method for staining electrophoresis gels for proteolytic activity of both serine and sulfhydryl enzymes is described. The gels are incubated in the presence of azocoll powder and then scanned.     

Staining for protease activity on polyacrylamide gels

A high-performance liquid chromatographic procedure has been developed for the detailed analysis of amino acids and related compounds in 10-μl samples of perilymph from the guinea pig cochlea (inner ear). The procedure employs an Aminco amino acid analyzer and combines the use of a single chromatographic column, lithium citrate buffers for elution, a change of column temperature, and fluorometric detection of o-phthaldialdehyde/2-mercaptoethanol adducts of primary amines. Sensitivity is about 0.2 pmol referenced to leucine. Fifty-four primary amine components are detectable in perilymph collected in relative silence. Twentynine compounds have been identified, and six are putative amino acid neurotransmitters. The present method provides new information about the chemical composition of perilymph and is suitable for the analysis of physiological fluids available only in volumes of several microliters.     

Caldesmon: anomalous electrophoretic behaviour in polyacrylamide gel

In SDS gels caldesmon (Mr = 140 kDa) and myosin light chain kinase (Mr = 130 kDa) migrate as a closely separated doublet. When glycerol is added to the gel caldesmon is characterized by an anomalous migration. In fact under this latter condition, the distance between caldesmon and myosin light chain kinase is enhanced by two-three times. The nature of putative caldesmon and myosin light chain kinase was confirmed by physicochemical, enzymatic and immunological methods.