A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
TL;DR: This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr with little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose.
About: This article is published in Analytical Biochemistry.The article was published on 1976-05-07. It has received 225085 citations till now. The article focuses on the topics: Bradford protein assay & Lowry protein assay.
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TL;DR: GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage, and Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
Abstract: We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
9,765 citations
TL;DR: The SRB assay provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening.
Abstract: We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute's disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.
9,019 citations
TL;DR: Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells that is suitable for creation of "on/off" situations for such genes in a reversible way.
Abstract: Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 microgram/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of "on/off" situations for such genes in a reversible way.
5,322 citations
TL;DR: In this article, a method for calculating accurate molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition, was presented, and the method was calibrated against 18 "normal" globular proteins.
5,077 citations
TL;DR: It is shown that micromolar concentrations of H2O2 can induce the expression and replication of HIV‐1 in a human T cell line and suggests that diverse agents thought to activate NF‐kappa B by distinct intracellular pathways might all act through a common mechanism involving the synthesis of ROI.
Abstract: Hydrogen peroxide and oxygen radicals are agents commonly produced during inflammatory processes. In this study, we show that micromolar concentrations of H2O2 can induce the expression and replication of HIV-1 in a human T cell line. The effect is mediated by the NF-kappa B transcription factor which is potently and rapidly activated by an H2O2 treatment of cells from its inactive cytoplasmic form. N-acetyl-L-cysteine (NAC), a well characterized antioxidant which counteracts the effects of reactive oxygen intermediates (ROI) in living cells, prevented the activation of NF-kappa B by H2O2. NAC and other thiol compounds also blocked the activation of NF-kappa B by cycloheximide, double-stranded RNA, calcium ionophore, TNF-alpha, active phorbol ester, interleukin-1, lipopolysaccharide and lectin. This suggests that diverse agents thought to activate NF-kappa B by distinct intracellular pathways might all act through a common mechanism involving the synthesis of ROI. ROI appear to serve as messengers mediating directly or indirectly the release of the inhibitory subunit I kappa B from NF-kappa B.
3,793 citations
References
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Journal Article•
TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
289,852 citations
TL;DR: An investigation of the biochemical changes following experimental liver injury felt the need of a simple, rapid, and accurate method for determining the protein fractions in small amounts of serum and began with Kingsley’s biuret procedure.
15,717 citations
TL;DR: This protein assay is described in which the sample is precipitated with trichloroacetic acid in the presence of sodium dodecylsulfate, filtered off on a Millipore membrane and stained with Amidoschwarz 10B, and its absorbance determined at 630 nm.
2,439 citations
TL;DR: A method has been developed to stain rapidly protein zones not only in standard but also in isoelectric focusing polyacrylamide gels that selectively visualizes the arginine- rich histones because of the solubility of the lysine-rich histones in PCA.
589 citations
Book•
01 Mar 1971
TL;DR: Handbook of biochemistry: selected data for molecular biology, Handbook of bi Chemistry: selectedData for molecular Biology, and so on.
Abstract: Handbook of biochemistry: selected data for molecular biology , Handbook of biochemistry: selected data for molecular biology , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی
555 citations