Showing papers on "Bradford protein assay published in 2002"

Food Protein Analysis: Quantitative Effects On Processing

Abstract: Fundamental techniques: Kjeld method quantitative amino acid analysis and combustion analysis. Copper binding methods: the alkaline copper reagent - biuret assay the Lowry method the bicinchoninic acid protein assay. Dye binding methods: the Udy methods the Bradford method - principles Bradford assay - applications. Immunological methods for protein speciation: immunological assay - general principles and the Agar diffusion assay speciation of meat proteins by enzyme-linked immunsorbent assay speciation of soya protein enzyme-linked immunoassay determination of trace protein allergens in foods. Protein nutrient value: biological and chemical tests for protein nutrient value effect of processing on protein nutrient value protein digestibility - corrected amino acid scores.

Spectrophotometric Determination of Protein Concentration

Abstract: This unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measured at 280 nm (A(280)) is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein (a(280)). Alternatively, absorbance measured at 205 nm (A(205)) is used to calculate the protein concentration. The A(280) and A(205) methods can be used to quantify total protein in crude lysates and purified or partially purified protein. A spectrofluorometer or a filter fluorometer can be used to measure the intrinsic fluorescence emission of a sample solution; this value is compared with the emissions from standard solutions to determine the sample concentration. The fluorescence emission method is used to quantify purified protein. This simple method is useful for dilute protein samples and can be completed in a short amount of time. There are two colorimetric methods: the Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to the protein of interest, and the Lowry method, which measures colorimetric reaction of tyrosyl residues in the protein sample.

Use of lab-on-a-chip technology for protein sizing and quantitation.

Abstract: The performance of the Agilent 2100 bioanalyzer, the first commercial lab-on-a-chip system, and the Protein 200 Plus LabChip kit is compared with conventional protein analysis techniques such as SDS-PAGE, Lowry, or Bradford. Lab-on-a-chip technology for protein analysis allows for the integration of electrophoretic separation, staining, destaining, and fluorescence detection into a single process, and for it to be combined with data analysis. The chip-based protein assay allows purity analysis, sizing, and relative quantitation based on internal standards or absolute quantitation based on user-defined standards. The chip-based protein analysis is comparable in sensitivity, sizing accuracy, and reproducibility to SDS-PAGE stained with standard Coomassie. Resolution and linear dynamic range are improved. Absolute quantitation accuracy and reproducibility is improved in comparison to SDS-PAGE and is comparable to batch-based quantitation methods such as Lowry and Bradford. The lab-on-a-chip system has several additional advantages over conventional SDS-PAGE including fast analysis times, reduced manual labor, automated data analysis, and good reproducibility. With such a system, the protein of interest can be tracked during the whole purification procedure, for example, from cell lysates through column fractions to purified proteins.

A quick assay for Na+-K+-AtPase specific activity.

Abstract: The method describes a simultaneous determination of inorganic phosphate (Pi) and protein content from a reaction mixture used for assay of adult rat cerebrocortical synaptosomal membrane Na+-K+-ATPase specific activity. The present method is more convenient, accurate and quicker compared to the existing methods for the determination of Na+-K+-ATPase activity. It also eliminates the possible errors in protein estimation by other classical methods in brain, which have a high lipid content.

Adaptation of commercial immunoassays to analysis of complex biological substances

Abstract: An Intracellular Adhesion Molecule I (ICAM-1) immunoassay from R and D Systems, and a Melanoma Inhibitory Activity (MIA) immunoassay from Roche Diagnostics were tested for accurate quantitation within complex biological substances such as cell lysates. Prior to assay, lysates of melanoma cells were treated with detergents to obtain soluble antigens. Maximum ICAM-1 and maximum MIA were detected after treatment using 0.8% Triton X-100. Two key aspects of assay accuracy were: 1) determining the dilutions of test sample that provided accurate quantitation (sample range), and 2) performing spiking experiments at these dilutions to determine absence or presence of a "matrix" effect due to biological complexity of the sample. A high degree of accuracy was found by diluting this particular cellular extract 50-fold prior to ICAM-1 assay, or only 5-fold prior to MIA assay. In addition, the bicinchoninic acid protein assay was analyzed to test the accuracy of protein quantitation of cellular lysates. Precision, limits of detection, and quantitation, robustness, linearity, and specificity also were tested for the immunoassays.

Purification of a zinc binding protein from Xylem of Citrus jambhiri

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.

Protein Quantitation: Bradford Method

Abstract: Protein quantitation according to the Bradford method. Keywords: protein quantitation; bradford; coomassie brilliant blue; calorimetric quantification

Modification of coomassic brilliant blue method and application to protein assay of tea liquor

Abstract: Modifications of Coomassie brilliant blue (CBB) method for proteins assay were made. The key factors affecting the results of tea liquor protein assay were studied, using bovine serum albumin (BSA) as external standard protein. The results showed that BSA-CBB complex and tea liquor proteins-CBB complex had similar ultra-visible (UV) absorption spectrum, and there were 4 peaks occurred at 205nm, 260nm, 305nm and 590nm, respectively, It also showed that the optimum conditions were 2.5~5.0 ml tea liquor, dyeing 30 min at 40℃, and 14.4ml dissolving solution. Protein concentrations in green tea and black tea solutions extracted at water: tea rate of 250ml:1.5g were 19.37μg/ml and 18.45μg/ml, with coefficient of variance of 0.54% and 1.05%, respectively.

Copper Iodide Staining of Proteins and Its Silver Enhancement

Abstract: Copper iodide staining and silver-enhancement is designed to quantify proteins adsorbed to solid surfaces such as nitrocellulose, nylon, polyvinylidene difluoride (PVDF), silica, cellulose, and polystyrene (1–5) and has important applications in Western blotting and thin layer chromatography (3,6). The binding of cupric ions to the backbone of proteins under alkaline conditions and their reduction to the cuprous state is the basis of several protein assays in solution including the biuret, Lowry, and bicinchoninic acid methods (1–3,7 and see Chapters 2–4). In the case of copper iodide staining, the protein binds copper iodide under highly alkaline conditions. This protein assay demonstrates sensitivity, speed, reversibility, low cost, and the lack of known interfering substances (including nucleic acid; refs. 4,5). Copper iodide staining is sufficiently sensitive to permit the quantification of proteins adsorbed to microtiter plates (5). The information is particularly useful for the quantitative interpretation of enzyme-linked immunosorbent assay (ELISA) and protein binding experiments. The precision of the determination of protein adsorbed to the microtiter plate by copper iodide staining is typically about 10–15%. The high sensitivity of copper iodide staining (about 40 pg/μL) may be increased several fold by a silver-enhancement procedure that allows the detection of protein down to about 10 pg/μL, which is more sensitive than common solution-based assays (7). The sensitivity of the assay can be increased by repeated applications of the protein on a membrane to concentrate it. Protein concentrations may be estimated from copper iodide staining from very dilute protein solutions or when only small amounts of a precious protein are available such as for the analysis of chromatography fractions.