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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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Patent
10 Jun 1976
TL;DR: A dye binding reagent for protein assay comprises Coomassie Brilliant Blue G-250 and an acid having a pKa of from 1 to 2 as discussed by the authors, which can be spectrophotometrically measured to quantitate even micrograms of protein.
Abstract: A dye binding reagent for protein assay comprises Coomassie Brilliant Blue G-250 and an acid having a pKa of from 1 to 2. Upon addition of the reagent to a protein-containing solution, the attendant color change can be spectrophotometrically measured to quantitate even micrograms of protein. Furthermore, the reagent is applicable to a wide range of proteins and requires only about 2 minutes for measurement. Addition of an alcohol such as ethanol to the reagent further improves sensitivity.

23 citations

Journal ArticleDOI
TL;DR: The conformation of extracellular matrix proteins can be altered by the argon laser at welding energies at low temperatures, even though no new covalent interactions were observed.
Abstract: Although the argon laser is used successfully to weld a number of different tissues, the underlying chemical and cellular mechanisms for this process are not precisely defined. Consequently, a biochemical model has been developed in vitro using the welldefined extracellular matrix from the murine Engelbreth-Holm-Swarm (EHS) sarcoma. Control and experimental samples of EHS basement membranes were irradiated with a Trimedyne argon laser at 500–3,000 Joules/cm2 at 0°C. The samples were diluted into cold phosphate-buffered saline and allowed to gel at 35°C. The time course of the gelation reaction was followed in a spectrophotometer at 360 nm. Irradiation reduced the absorbance 7.5–15% compared to controls and was independent of the dilution over a 10-fold range. Gelation was also measured by determining the amount of protein by the Bradford assay that could be collected by centrifugation at 10,000g for 10 minutes. Argonirradiated samples had 30–40% less protein in the precipitate than the controls. The addition of 5 mM beta-mercaptoethanol to the EHS extract blocked the effect of the laser on the gelation reaction. In addition, when gelation was carried out in the absence of calcium and magnesium, there were no differences between laser-treated samples and controls. The basement membrane proteins were separated by electrophoresis through polyacrylamide gels under denaturing plus reducing or denaturing and non-reducing conditions. No differences in the polypeptide composition were noted between irradiated and control samples using either Coomassie- or silver-staining techniques. The irradiated and control samples were also analyzed on 1.5% agarose gels in 10 mm Tris-HCl, pH 8 containing 0.1% SDS. Both samples produced a single Coomassie-positive band, but the control migrated at 0.5 and the irradiated sample at 0.6. In sum, the conformation of extracellular matrix proteins can be altered by the argon laser at welding energies at low temperatures, even though no new covalent interactions were observed.

23 citations

Journal ArticleDOI
TL;DR: In this paper, the authors proposed a method for protein determination based on the amino acid composition of IUPs, which is called order-promoting amino acids (OPA-AAs).

23 citations

Journal ArticleDOI
TL;DR: Pressure cycling technology uses alternating cycles of high and low hydrostatic pressure to effectively induce the lysis of cells and tissues in preparation for 2DGE and other analytical or preparative methods.

23 citations

Journal ArticleDOI
TL;DR: This study delineates the variability in absolute quantification of polymorphic CYP2D6 drug-metabolizing enzyme and compares immunoblot and nano liquid chromatography coupled to mass spectrometry (nano-LC/MS) methods in identification and relative quantification, and suggests that nano- LC/MS not only facilitates P450 quantitation but also provides genotypic information.
Abstract: Accurate quantification of cytochrome P450 (P450) protein contents is essential for reliable assessment of drug safety, including the prediction of in vivo clearance from in vitro metabolism data, which may be hampered by the use of uncharacterized standards and existence of unknown allelic isozymes. Therefore, this study aimed to delineate the variability in absolute quantification of polymorphic CYP2D6 drug-metabolizing enzyme and compare immunoblot and nano liquid chromatography coupled to mass spectrometry (nano-LC/MS) methods in identification and relative quantification of CYP2D6.1 and CYP2D6.2 allelic isozymes. Holoprotein content of in-house purified CYP2D6 isozymes was determined according to carbon monoxide difference spectrum, and total protein was quantified with bicinchoninic acid protein assay. Holoprotein/total CYP2D6 protein ratio was markedly higher for purified CYP2D6.1 (71.0%) than that calculated for CYP2D6.1 Supersomes (35.5%), resulting in distinct linear calibration range (0.05–0.50 versus 0.025–0.25 pmol) that was determined by densitometric analysis of immunoblot bands. Likewise, purified CYP2D6.2 and CYP2D6.10 and the CYP2D6.10 Supersomes all showed different holoprotein/total CYP2D6 protein ratios and distinct immunoblot linear calibration ranges. In contrast to immunoblot, nano-LC/MS readily distinguished CYP2D6.2 (R296C and S486T) from CYP2D6.1 by isoform-specific proteolytic peptides that contain the altered amino acid residues. In addition, relative quantitation of the two allelic isozymes was successfully achieved with label-free protein quantification, consistent with the nominated ratio. Because immunoblot and nano-LC/MS analyses measure total P450 protein (holoprotein and apoprotein) in a sample, complete understanding of holoprotein and apoprotein contents in P450 standards is desired toward reliable quantification. Our data also suggest that nano-LC/MS not only facilitates P450 quantitation but also provides genotypic information.

23 citations


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