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Bradford protein assay

About: Bradford protein assay is a research topic. Over the lifetime, 635 publications have been published within this topic receiving 239107 citations.


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Journal ArticleDOI
TL;DR: The Coomassie brilliant blue assay for the determination of protein has been extended to rapidly and conveniently measure the protein concentration of cells growing in culture in a 96-well microtiter format.

5 citations

Journal ArticleDOI
TL;DR: In this paper, a Zn-binding protein (CVZBP) was found to be anionic, requiring 0.43 N NaCl for elution from quaternary aminoethyl Sepharose, and further purified by sodium dodecyl sulfate-polyacrylamide gel-stage electrophoresis.
Abstract: sporamin ABSTRACT. Zinc in xylem and phloem of the citrus rootstock, rough lemon (Citrus jambhiri (L.)) was associated with a Zn- binding protein, designated citrus vascular Zn-binding protein (CVZBP). The apparent molecular mass of the CVZBP was 19.5 kDa after nondenaturing size exclusion chromatography and 21.8 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ion exchange chromatography demonstrated that CVZBP was anionic, requiring 0.43 N NaCl for elution from quaternary aminoethyl Sepharose. Antiserum to the protein cross-reacted more with total protein extracts from leaf midveins than with total protein from the rest of the leaf lamina, further suggesting a vascular location of the Zn-binding protein. Quantitative analysis indicated that ≈2 to 3 mol of Zn were associated with 1 mol of native protein. Binding studies with the partially purified CVZBP demonstrated a capacity to bind several divalent cations: Cd, Ni, Pb, and Zn. Reaction with Ellman's reagent suggested that the protein has significant sulfhydryl group content that may be involved in metal binding. N-terminal sequencing demonstrates identity with papaya latex trypsin inhibitor, sporamin, or other Kunitz soybean proteinase inhibitors. samples were collected from healthy or blight affected trees of Citrus jambhiri grown in a quarantine greenhouse facility on the campus of University of Arizona, Tucson, during 1996. Xylem evacuate was collected by applying 4 mL of 10 mM Tris-Cl, pH 7.4 to one end of a 10 cm root piece >1 cm diameter while a vacuum was pulled at the other end. Bark was removed from the root piece down to the cambium before evacuation. Before purification, xylem evacuate was stored at -80 °C. Phloem tissue was sampled as a bark patch taken 15 to 30 cm above the soil line. Contaminants on the bark surface were removed by scraping away the brown-green outer layer to a 5 × 10 cm rectangle of clean whitish phloem tissue. The patch was scored through the scraped bark to the wood and separated from the tree at the cambial layer. The samples were taken from convex surfaces of healthy and blight-affected trees in active growth. ISOLATION AND PURIFICATION OF CVZBP. To quantitate recov- ery of CVZBP, three independent purifications were performed. Three separate xylem evacuates (Derrick et al., 1990) were lyophilized and adjusted to 1 mL each with deionized water. Xylem evacuate was fractionated by ion exchange chromatogra- phy (IEC) (Taylor et al., 1996) over quaternary aminoethyl Sepharose (QAE) (Pharmacia, Piscataway, N.J.). Total Zn was determined for aliquots of each fraction by determination of A213.9 by atomic absorption spectrometry (model 3100, Perkin-Elmer, Shelton, Conn.) and for 254 nm absorbance (A254) by ultraviolet (UV)- spectrophotometry. Fractions with elevations of A254 and coincident elevated Zn were pooled and the volume reduced in a centrifugal vacuum concentrator (model RC 10. 10; Jouan, Win- chester, Va.). Concentrated Zn-containing eluant from QAE chromatography was separated using a 2.5 × 14 cm gel filtration (GF) column of Bio-Gel P-30 (Bio-Rad, Hercules, Calif.) in 10 mM Tris-Cl buffer, pH 8.0 (4 °C) in 3 mL fractions. Fractions within the peaks of A254 and elevated Zn content were pooled, trichloroacetic acid precipitated (Deutscher, 1990), and further purified by sodium dodecyl sulfate-polyacrylamide gel electro- phoresis (SDS-PAGE) (Laemmli, 1970). For all isolation steps, total protein was determined at A595 with Quantigold (Diversified Biotech, Newton Centre, Mass.) or with the Bradford assay (Bio- Rad), depending upon protein concentration with each stage of purification.

5 citations

Book ChapterDOI
01 Jan 1981
TL;DR: The Lowry assay depends on the complexing of copper with tyrosine and tryptophan residues, whereas the Bradford method measures the binding of Coomassie Brilliant Blue G-250 to protein.
Abstract: Publisher Summary The Lowry and Bradford assays for protein determination provide relatively simple methods for calculating specific activities by standardizing enzyme activities to protein content. Both assays are colorimetric. The Lowry assay depends on the complexing of copper with tyrosine and tryptophan residues, whereas the Bradford method measures the binding of Coomassie Brilliant Blue G-250 to protein. The protocols are modified specifically for protein determination in Triton lysates but may be altered for different conditions by simply replacing Triton with the appropriate agent. The standards may be plotted on a linear graph of concentration versus absorbance, and unknown concentrations may then be read directly from the graph. Alternatively, a straight line may be fit to the standard data by the method of least squares and the formula generated may be used to calculate unknown concentrations. This latter method is especially convenient for use with a hand-held programmable calculator. The blank cuvette should be rinsed approximately every 10 min to avoid progressive staining of the cuvette.

5 citations

Patent
11 May 2011
TL;DR: In this article, a method for analyzing protein content in 2-keto-L-gulonic acid, which comprises the following steps: adopting an optimized Coomassie brilliant blue quantitative protein method (a Bradford method), preparing phosphate buffer solution, bovine serum protein standard solution, adding the Coomenie Brilliant blue G-250 staining agent, drawing a standard curve according to absorbency added values after the Coomasserie brilliantblue is combined with the protein, and calculating the protein content.
Abstract: The invention relates to a method for analyzing protein content in 2-keto-L-gulonic acid, which comprises the following steps: adopting an optimized Coomassie brilliant blue quantitative protein method (a Bradford method), preparing phosphate buffer solution, bovine serum protein standard solution and Coomassie brilliant blue G-250 staining agent, adding the Coomassie brilliant blue G-250 stainingagent into the bovine serum protein standard solution, drawing a standard curve according to absorbency added values after the Coomassie brilliant blue is combined with the protein, then adding the 2-keto-L-gulonic acid into a Coomassie brilliant blue reagent, and calculating the protein content in the 2-keto-L-gulonic acid according to the absorbency added values and the slope coefficient of a protein concentration standard curve. The analyzing method is simple, convenient and sensitive, has the advantages of good stability, high accuracy, good repeatability and low requirement on detection equipment, is quite suitable for analyzing and detecting the protein content in the 2-keto-L-gulonic acid in the industrial production.

5 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
20238
202212
202127
202021
201919
201822