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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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TL;DR: In this paper, the resonance Rayleigh scattering (RRS) spectrum of 4-azochromotropic acid phenylfluorone has a peak at 337nm, and the RRS intensity is enhanced by small amounts of protein due to the binding interaction of protein and the dye.
60 citations
TL;DR: It is concluded that standardization of Cry1Ab production and quantification by SDS-PAGE/densitometry may improve data consistency in monitoring efforts to identify changes in insect susceptibility toCry1Ab.
Abstract: Standardization of toxin preparations derived from Bacillus thuringiensis (Berliner) used in laboratory bioassays is critical for accurately assessing possible changes in the susceptibility of field populations of target pests. Different methods were evaluated to quantify Cry1Ab, the toxin expressed by 80% of the commercially available transgenic maize that targets the European corn borer, Ostrinia nubilalis (Hubner). We compared three methods of quantification on three different toxin preparations from independent sources: enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (SDS-PAGE/densitometry), and the Bradford assay for total protein. The results were compared to those obtained by immunoblot analysis and with the results of toxin bioassays against susceptible laboratory colonies of O. nubilalis. The Bradford method resulted in statistically higher estimates than either ELISA or SDS-PAGE/densitometry but also provided the lowest coefficients of variation (CVs) for estimates of the Cry1Ab concentration (from 2.4 to 5.4%). The CV of estimates obtained by ELISA ranged from 12.8 to 26.5%, whereas the CV of estimates obtained by SDS-PAGE/densitometry ranged from 0.2 to 15.4%. We standardized toxin concentration by using SDS-PAGE/densitometry, which is the only method specific for the 65-kDa Cry1Ab protein and is not confounded by impurities detected by ELISA and Bradford assay for total protein. Bioassays with standardized Cry1Ab preparations based on SDS-PAGE/densitometry showed no significant differences in LC50 values, although there were significant differences in growth inhibition for two of the three Cry1Ab preparations. However, the variation in larval weight caused by toxin source was only 4% of the total variation, and we conclude that standardization of Cry1Ab production and quantification by SDS-PAGE/densitometry may improve data consistency in monitoring efforts to identify changes in insect susceptibility to Cry1Ab.
59 citations
TL;DR: This assay compares favorably to other assay procedures in terms of rapidity, sensitivity, expense, and lack of interference by many laboratory reagents, although like the others it suffers from the drawback of differences in response of different proteins, which is inherent in dye-binding assays.
59 citations
TL;DR: A method that is capable of accurately measuring low amounts of a protein in the presence of very high levels of lipid is described, developed from the amido black 10 B methods and incorporates several critical modifications that enable an assay to be performed with lipid-containing samples without any interference.
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.
59 citations
TL;DR: It is found that sucrose not only could increase the rate of protein recovery after 1 h acetone precipitation, but also did not interfere with Bradford assay, so a method for rapid protein quantitation in protein drugs even if they contained interfering substances is developed.
58 citations