Showing papers on "Bradford protein assay published in 1990"

Molecular surgery of the basement membrane by the argon laser.

Abstract: Although the argon laser is used successfully to weld a number of different tissues, the underlying chemical and cellular mechanisms for this process are not precisely defined. Consequently, a biochemical model has been developed in vitro using the welldefined extracellular matrix from the murine Engelbreth-Holm-Swarm (EHS) sarcoma. Control and experimental samples of EHS basement membranes were irradiated with a Trimedyne argon laser at 500–3,000 Joules/cm2 at 0°C. The samples were diluted into cold phosphate-buffered saline and allowed to gel at 35°C. The time course of the gelation reaction was followed in a spectrophotometer at 360 nm. Irradiation reduced the absorbance 7.5–15% compared to controls and was independent of the dilution over a 10-fold range. Gelation was also measured by determining the amount of protein by the Bradford assay that could be collected by centrifugation at 10,000g for 10 minutes. Argonirradiated samples had 30–40% less protein in the precipitate than the controls. The addition of 5 mM beta-mercaptoethanol to the EHS extract blocked the effect of the laser on the gelation reaction. In addition, when gelation was carried out in the absence of calcium and magnesium, there were no differences between laser-treated samples and controls. The basement membrane proteins were separated by electrophoresis through polyacrylamide gels under denaturing plus reducing or denaturing and non-reducing conditions. No differences in the polypeptide composition were noted between irradiated and control samples using either Coomassie- or silver-staining techniques. The irradiated and control samples were also analyzed on 1.5% agarose gels in 10 mm Tris-HCl, pH 8 containing 0.1% SDS. Both samples produced a single Coomassie-positive band, but the control migrated at 0.5 and the irradiated sample at 0.6. In sum, the conformation of extracellular matrix proteins can be altered by the argon laser at welding energies at low temperatures, even though no new covalent interactions were observed.

Composition for determining trace amount of protein

Abstract: A new and improved method and composition for determining the presence and concentration of low to trace amounts of proteins, such as albumin, in a test sample, such as urine. The method includes using a reagent composition capable of interacting with low trace amounts of proteins to produce a visually or instrumentally detectable and/or measurable response. The method can be used in wet assays or in dry test strip assays, wherein the reagent composition is incorporating into a carrier matrix. The new and improved reagent composition, comprising a dye, such as a polyhydroxybenzenesulfonephthalein-type indicator, like pyrocatechol violet; a tungstate, such as ammonium tungstate; and, if necessary, a suitable buffer, is incorporated into the carrier matrix to provide sufficient sensitivity to low to trace protein levels and sufficient color resolution between low to trace protein levels, thereby affording an accurate and trustworthy protein assay of test samples having a low protein concentration.

[24] Purification of exocellular superoxide dismutases

Abstract: Publisher Summary This chapter discusses a technology used for conducting comparative exocellular forms of the enzyme extracellular superoxide dismutase (EC-SOD) therapy of the physiological diseases of the mutants. Human EC-SOD is a tetrameric glycoprotein, whose molecular weight is 135,000. The chapter describes purification procedures for selecting proteins of this nature. In SOD assay, the spectrophotometric method of Misra and Fridovich is used. The method is based on the inhibition by the enzyme of the spontaneous, alkali-catalyzed autoxidation of epinephrine to norepinephrine. In protein assay, the spectrophotometric method of Murphy and Kies is used. The course of dialysis to remove tetraborate is monitored by a colorimetric assay. A sample of the dialysate (0.9 ml) is mixed with 0.1 ml of 1% silver nitrate and incubated at room temperature for about 10 minutes or until the absorbancy at 500 nm becomes constant. The extinction coefficient of tetraborate is 0.255. The assay detects as little as 10–100 μ g sodium tetraborate per milliliter. The purification procedures are based on the assumption that all EC-SODs are large, highly glycosylated proteins. The literature on highly glycosylated proteins indicates that their properties resemble that of polysaccharides and sugars. Solubility and resistance to denaturation in organic solvents is one feature of highly glycosylated proteins exhibited by EC-SODs. They are soluble and remain active in 80–95% ethanol. The yeast enzyme is also soluble and fully active after extraction in 80% phenol. Furthermore, unlike ordinary protein, EC-SODs are soluble in aqueous saturated ammonium sulfate.

Protein assay method

Abstract: A method for protein assay is a modified Lowry method in which a Step 1 reaction between a copper-containing alkaline solution and a protein solution takes place at high alkali concentration. The Step 2 reaction between the products of Step 1 and Folin reagent commences at a pH of between 11 and 12 in order to produce rapidly the coloured species on the basis of which a maximum optical density is reached in a relatively short time and remains constant for a sufficient period to enable the required number of optical density determinations to be made.

Assays for superoxide dismutase based on autoxidation of hematoxylin.

Abstract: Publisher Summary This chapter discusses the assays for superoxide dismutase (SOD) based on the autoxidation of hematoxylin. The assay is based on the transformation of hematoxylin to its two-electron-oxidized product, hematein. The assay is sensitive and simple. The reaction product, hemarein, is relatively stable and has an absorption maximum and extinction coefficient similar to those of the commonly used O 2 – scavenger, cytochrome c . The reaction is inhibited by SOD and can be performed in the physiological pH range of 6.8–7.8, where SOD inhibits the reaction 90–95%. In addition, at pH values above 8.1, the autoxidation reaction is accelerated by added SOD, and the reaction becomes a positive assay for the enzyme, similar in principle to SOD assays based on the photochemical and enzymatic oxidation of dianisidine. The activity of purified SOD is determined by the xanthine oxidase cytochrome c method. Xanthine oxidase is isolated from unpasteurized cream. General protein is determined by the Bradford assay using bovine serum albumin (BSA) as a standard.

Protein assay method and kit

Abstract: A method for protein assay is a modified Lowry method in which a Step 1 reaction between a copper-containing alkaline solution and a protein solution takes place at high alkali concentration. The Step 2 reaction between the products of Step 1 and Folin reagent commences at a pH of between 11 and 12 in order to produce rapidly the coloured species on the basis of which a maximum optical density is reached in a relatively short time and remains constant for a sufficient period to enable the required number of optical density determinations to be made.