Showing papers on "Bradford protein assay published in 1997"

Enhancement of Rayleigh Light Scattering of Acid Chrome Blue K by Proteins and Protein Assay by the Scattering Technique

Abstract: Rayleigh light scattering of Acid Chrome Blue K (ACBK) is enhanced greatly by proteins. Based on this, a method for protein assay in aqueous solution was developed. This assay matches the sensitivity of the colorimetric dye-binding method with a linear range of 0.136–10.88 µg ml - 1 . The measurements can be made easily on a common fluorimeter. The reaction between ACBK and proteins is completed in 2 min and the scattered light signal is stable for at least 3 h. Protein-to-protein variability is encountered in this method as in many other protein assays. There is little or no interference from amino acids, most metal ions and complexing agents (e.g., EDTA). Interferences from salts, urea and detergents can be minimized by dilution.

Quantification of interferences in the direct measurement of proteins in wines from the Champagne region using the Bradford method

Abstract: At this time, the Bradford method is the most commonly used method in enology for direct protein quantification because of its rapidity and its reproducibility. This paper considers interferences which may falsify the estimation of protein in champagne Pinot noir and Chardonnay wines. Ultrafiltration with 10 kDa MWCO membrane shows that nonprotein compounds largely react with the Coomassie Brilliant Blue (CBB) and can produce an A 595 nm equal to 30% to 90% of the initial value. Ethanol and exogenous and endogenous phenolic compounds are principally responsible for these interferences. They act indirectly favoring the neutral form of the dye. However, interferences from ethanol and phenolics are not additive. Furthermore, the interaction is negative. Gelatins used in enology react 50 times less than BSA with the dye reagent. Both bentonite and vegetable charcoal treatments partially eliminate proteins and phenolics. The ratios A 520 nm /A 595 nm (eq. BSA estimated by the direct Bradford method) increases on increasing bentonite fining and decreases on charcoal treatment. For these reasons, the deproteinization effects of enological treatments are, at present, very difficult to estimate with direct measurement. A modified Bradford method involving of A 595 nm before and after ultrafiltration (3 and 10 kDa MWCO) is proposed as a more accurate measure of wine protein content.

Measurement of Protein Content in Fruit Juices, Wine and Plant Extracts in the Presence of Endogenous Organic Compounds

Abstract: Measurement of soluble protein in wines, fruit juices and enzyme extracts by the standard Coomassie blue assay gave results that did not agree with protein content calculated from samples and standards on silver-stained SDS-PAGE gels. A simple one-step change has improved the quantitative measurement of protein The acidic pH of wines and fruit juices, and acidic to neutral pH of vegetable extracts favour hydrogen bonding of some organic compounds to protein. In a modified Coomassie blue assay the samples were made alkaline to reduce hydrogen bonding of protein to phenolics, fats and their breakdown products. Electrostatic binding to the positively charged guanidino group of arginine, the amino acid responsible for the colour change when protein interacts with Coomassie blue dye, was also reduced. On addition of Coomassie blue dye (pH 0.70) there may be competition for binding to protein by organic compounds and dye, rather than a strengthening of the already hydrogen-bound compounds to protein. Protein content measured by the standard Coomassie blue assay and by alkaline treatment of the samples differed significantly. Results from the ‘alkali assay’ agreed quantitatively with protein estimates from silver-stained SDS-PAGE gels.

Microbial Physiology and Biochemistry Laboratory: A Quantitative Approach

Abstract: Introduction Acknowledgements Laboratory Rules and Safety Experiment 1: An exercise in pipetting and the Beer-Lambert Law Experiment 2: Bacterial growth curve Experiment 3: Bioassay for micotinic acid Experiment 4: The effect of environment on growth Experiment 5: Lactic acid production by lactic acid bacteria Experiment 6: Assay of protein and RNA in whole cells grown at different growth rates Experiment 7: Analysis of diauxic growth Experiment 8: Assay of amylase and protease secreted by Bacillus Subtilis Experiment 9: Concentration of amylase from Bacillus Subtilis by ammonium sulfate precipitation and separation from protease by affinity purification Experiment 10: Ion-exchange chromatography of amylase Experiment 11: Induction of alkaline phosphatase and the determination of its cellular cation in Escherichia coli Experiment 12: Assay of threonine deaminase: Determination of KM and Vmax Experiment 13: Assaying fructose-1,6-bisphosphatase in Saccharomyces cerevisiae Experiment 14: Purification of glucose-6-phosphate dehydrogenase Experiment 15: How E. coli adapts to anaerobiosis: Nitrate reductase Experiment 17: Chemotaxis of Pseudomonas aeruginosa Experiment 18: Phototasix of Rhodospirllum centenum Experiment 19: Light production by Phorobacterium phosphoreum Appendix A: Analysis of experimental data: How to do simple dilutions Guidelines for writing laboratory reports Appendix B: Laboratory supplies and equipment Appendix C: Media Buffers Solutions Appendix D: RNA assay using orcinol Lowry protein assay Bradford Protein assay Assay for *a-amylase using starch conjugated to a dye Appendix E: The Beer-Lambert law Relative absorbancies Cell density vs. Klett units Appendix F: Quantitative problems Appendix G: Solutions to problems in Appendix F Appendix H: Determining the amount of inorganic phosphate that limits the growth yields of E. coli, Factors that affect the relative amounts of saturated unsaturated, and branched chain fatty acids Measuring the KM and Vmax of fructose-1,6-bisphosphatase and the inhibition by AMP Assaying glycolytic enzymes and fructose-1,6-bisphosphatase in yeast grown on glucose or ethanol Regulation of phosphofructokinase activity Further experiments and demonstrations with Photobacterium

Characterization of Enzyme Activity, Protein Content, and Thiol Groups in Immobilized Enzymes

Abstract: Immobilization is designed to restrict the freedom of movement of an enzyme and, in doing so, places limitations on the enzyme and the biotransformation catalyzed by the enzyme (see Chapter 1). In practical terms, this often means that the normal procedures used for assay of the soluble enzyme activity, protein content, and so on, must be redesigned to accomodate the presence of the support material. In general, problems are likely to be experienced when the active agent in an assay procedure binds dnectly to the enzyme and measurement is based on the enzyme-reagent complex. Support materials are likely to cause interference in such procedures. Determination of protein content in immobilized enzymes can be estimated by direct and indirect methods (Z-3). The indirect approach involves protein determination before and after immobilization, and is less convenient and less accurate when extensive washing of support material is required to remove unbound protein. The direct method involves reaction of the immobilized protein with protein assay reagent, and it is essential that the products of the reaction are released into solution so they can be separated from the support material and assayed without interference (45). For example, protein assay based on Coomassie Blue dye binding is not suitable for immobilized enzyme because the dye binds to the immobilized protein (6) and in effect becomes immobilized to the support material. The Lowry protein assay is more appropriate for immobilized enzymes because the colored product is released into solution and can be separated from the mnnobihzed enzyme by filtration or centrifugation. An alternative method involves use of polyethylene glycol to diminish light scattering of agarose beads and subsequent direct spectrophotometric measurement of bound protein at 280 nm (3).

Determination of the Reaction Product of Glutaraldehyde and Amine Based on the Binding Ability of Coomassie Brilliant Blue

Abstract: Coomassie Brilliant Blue G-250 (CBBG) was bound to the reaction products of glutaraldehyde with various compounds containing amino group. The reaction products of acetaldehyde and malonaldehyde with the amines did not bind with CBBG, indicating that the binding was specific for the product of glutaraldehyde. The dye binding products rapidly formed in alkaline pH. Gel filtration chromatography indicated that a reaction product of more than 2 kDa has a dye- binding ability. The reaction product of glutaraldehyde with 6-aminohexanoic acid was separated with SP-Sephadex. CBBG was bound to the first eluted fraction from the column with highest affinity. The absorbance at 595nm by CBBG binding was linearly related to this lyophilized fraction at a concentration of up to 250μg/ml, suggesting its applicability to the determination of the reaction product of glutaraldehyde and amine. The present method should contribute to our understanding of the chemistry of glutaraldehyde associated with diverse fields, such as enzyme technology and biochemistry.