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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. 相似文献
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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. 相似文献
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A procedure for the direct staining of argininosuccinate lyase activity in polyacrylamide gel is described. The method was based on coupling one of the enzymatic products fumarate with fumarase and malic enzyme catalyzed reactions. Fumarate was first converted to L-malate by fumarase. Malic enzyme then catalyzed the oxidative decarboxylation of L-malate to give CO2 and pyruvate with concomitant reduction of NADP+ to NADPH. Finally the reducing power of NADPH was coupled to phenazine methosulfate and in turn to nitroblue tetrazolium yielding a deeply colored insoluble formazan which may be quantitized or semiquantitized by densitometer. 相似文献
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Maizel JV 《Trends in biochemical sciences》2000,25(12):1318-592
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Four of the membrane proteins from Acholeplasma laidlawii that are soluble in the nonionic detergent Tween 20 have been purified by preparative electrophoretic techniques utilizing different supporting media. The last purification step for two of the major proteins was a preparative polyacrylamide gel electrophoresis performed in the absence of any detergent. The proteins were recovered by continuous elution. The purity of the fractions was examined by analytical polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. Two of the minor proteins were purified by dextran gel electrophoresis as the final step, which was also performed in a detergent-free buffer. The separation was followed by scanning the dextran gel in ultraviolet light. The proteins were recovered by slicing the gel and degrading the gel slices with dextranase. The homogeneity of the fractions was checked by electroimmunoassay. 相似文献
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The velocities of single stranded DNA molecules in denaturing polyacrylamide gels during symmetric and asymmetric field inversion were measured at different pulse times and gel concentrations. Under the conditions chosen in our study, pulse times as short as a few milliseconds lead to a retardation of DNA molecules larger than 400 bases. We found that a field inversion with an electric field in the forward direction of about double the strength of that applied in the backward direction is a good compromise between the degree of retardation, the temperature control requirements and the run time of the gel. 相似文献
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The state of adsorbed water in a dextran gel has been investigated by near-infrared and gravimetric-adsorption techniques. Water-vapor adsorption (desorption) isotherms at three temperatures are reported. The calculated sorption heats are found to be markedly temperature-dependent as well as dependent on the coverage. The near-infrared spectrum (4650–9000 cm-1) is reported, together with tentative assignments. The H2O combination (ν + δ) band at 5184 cm-1 has been examined as a function of relative humidity. The line-shapes of this band have been analyzed by a recently established, Fourier-inversion technique, and information on the microdynamics of the adsorbed water molecules has been resolved on the picosecond time-scale. At low and intermediate degrees of hydration, reorientational jumps take place with periods from four to six times longer than those for free water. The onset of saturation is then accompanied by the sudden removal of the reorientational jumps. A comparison of microdynamical and thermodynamic data indicates the hydration mechanism to be highly cooperative at all relative humidities. 相似文献
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A simplified procedure for the determination of the glutamate chain lengths of labeled and endogenous tissue folate is described. Pteroylpoly-γ-glutamates in tissue extracts were reductively cleaved at the C,9N,10 bond to p-aminobenzoylpolyglutamates, which were converted to azo dyes by coupling their diazonium salts with naphthylethylene diamine. The azo dyes were well resolved, according to glutamate chain length, by gel chromatography on Bio-Gel P4. Unlabeled tissue folates were detected by the absorbance of their azo dye derivatives. The major endogenous pteroylpolyglutamate in rat liver, identified colorimetrically using 0,5 g tissue, was the pentaglutamate. The major labeled folates in Lactobacillus casei and Streptococcus faecalis, after incubating these bacteria with labeled folic acid, were identified as the octa- and tetraglutamates, respectively. Reductive cleavage of 10-formylfolate and 5.10-methenyltetrahydrofolate resulted in a mixture of N-substituted and unsubstituted p-amino-benzoylpolyglutamates. Methods are described for the complete cleavage of these formyl derivatives to unsubstituted p-aminobenzoylpolyglutamates. 相似文献
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Cylindrical polyacrylamide gels were automatically analyzed for radioactive 14C using a commercially available radiochromatogram scanner. This method was capable of detecting peaks of [14C]protein containing 50 000--1 000 000 cpm. Under optimal conditions, gels could be scanned at a rate of 1 cm per min, allowing for analysis of 20--30 gels per day. 相似文献
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A procedure was developed for a rapid staining of proteins in polyacrylamide gels with tannic acid and the extraction of enzymatic activity from the gels. Lysozyme and Taka-amylase A were stained with tannic acid and localized on pH 4.3, and 8.0 and 9.5 gels, respectively. After the gels were rinsed in buffer solutions, the activities of the enzymes were recovered in good yield from the gels. The use of these techniques is discussed. 相似文献
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Isoelectric focusing in ampholytes of pH 6–8 range has been carried out in polyacrylamide gels using ammonia buffer at pH 10.0 and acetate buffer at pH 4.0 for the cathode and anode solutions, respectively. This system requires low voltages but compares well with isoelectric focusing using strong acid and strong base electrode solutions. The advantages of this method are the less extreme pH's in the electrode solutions and lower resistance in the neutral region of the pH gradients. 相似文献