Showing papers on "Bradford protein assay published in 1989"

Measuring plant protein with the Bradford assay : 1. Evaluation and standard method.

Abstract: The suitability of the Bradford protein assay for measuring plant protein was evaluated and a standard method developed. The assay involves extraction of dried, fresh, or frozen plant material in 0.1 NaOH for 30 min. Replicate 100-μl aliquots of centrifuged supernatant are assayed with 5 ml Bio-Rad Bradford dye reagent (Coomassie brilliant blue G-250) diluted 1:4 and containing 3 mg/ml soluble polyvinylpyrollidone. Absorbance at 595 nm is recorded after 15 min against an NaOH blank. Samples are calibrated against a ribulose 1,5-diphosphate carboxylase-oxygenase standard in NaOH. Procedures for plant preparation, extraction stability, the effects of phenol removal and quinone formation, and assay recovery are evaluated. Assay absorbance stability and techniques for increasing absorbance stability are reported. Changes in protein quality are briefly discussed.

Sensitive protein assay in presence of high levels of lipid.

Abstract: Publisher Summary This chapter describes a method that is capable of accurately measuring low amounts of a protein in the presence of very high levels of lipid. This procedure was developed from the amido black 10 B methods of Schaffner and Weissmann and Newman et al. and incorporates several critical modifications that enable an assay to be performed with lipid-containing samples without any interference. One approach has been to remove an interfering lipid by extraction with organic solvents. However, because certain proteins display a limited solubility in such solvents, this strategy often fails. Another widely used approach involves the inclusion of sodium dodecyl sulfate (SDS) in a modified Lowry procedure to reduce lipid (and detergent) interference. As oxidized lipid continues to react to produce a substantial amount of color in the Lowry assay and as most lipid samples are partially oxidized, this procedure is not suitable for the accurate measurements of a protein in samples containing excess of lipid.

Development of a human milk protein standard

Abstract: . Accurate quantitation of the protein content of human milk (HM) is clinically important for both determining protein and energy intakes of HM-fed infants. Protein can be determined by Kjeldahl analysis or colorimetric assays. Colorimetric assays are rapid and convenient, but usually overestimate the protein content when compared to Kjeldahl protein (KP). In this study a protein standard based on human milk protein (HMPS) was isolated by size exclusion column chromatography. Purity and composition of the standard was determined by gel electrophoresis, Kjeldahl and amino acid analyses. The protein content of 20 mature milk samples was determined by the Lowry, BCA and BioRad colorimetric assays, using bovine serum albumin, human serum albumin/IgG, and HMPS as standards. These results were compared with KP results using Student's f-test. All colorimetric assays overestimated milk protein content; however, the Lowry assay gave the lowest protein levels, with our protein standard yielding values closest to the KP value.

Composition and method of assaying for trace amounts of proteins

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 to 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.

Characterization of protein binding to a nitrocellulose membrane

Abstract: Proteins bound to a nitrocellulose (NC) membrane tightly and quantitatively until the NC membrane was saturated with the proteins. The binding of proteins to the NC membrane was characterized by using twenty proteins having different molecular weight and isoelectric point as follows: 1) hydrophobic interactions between the protein and the NC membrane matrices may play a major role in the protein binding, and sugars, amino acids, DNA, nucleotides, neutral salts, or glycerol in the protein solution did not interfere with the protein binding. 2) The number of the protein molecule bound to the NC membrane was in the range from 1.13 to 1.98nmol/cm2 except for a few small and strongly charged proteins. 3) Non-ionic detergents such as Tween 20, Triton X-100, and Nonidet P-40 strongly interfered with the protein binding to the NC membrane in a concentration dependent manner. 4) Proteins could be extracted from the NC membrane with high yield by using a low concentration of the non-ionic detergent. These enables us to assay the proteins with high sensitivity and reproducibility. Furthermore, it may be possible to determine the amino acid composition and sequence with small amount of the proteins after the assay.