Quantitation of protein.
TL;DR: This chapter discusses various methods of estimating protein concentration as defined by the difference in energy between the orbital of the unexcited electron and a higher energy orbital.
Abstract: Publisher Summary This chapter discusses various methods of estimating protein concentration. Absorption spectroscopy involves the absorption of a photon by an electron. Only those photons with a certain energy level can be absorbed as defined by the difference in energy between the orbital of the unexcited electron and a higher energy orbital. The peptide bond absorbs photons below 210 nm. Because of the large number of peptide bonds in a protein, this is a highly sensitive area of the protein spectrum. Although protein conformation and some absorption by tryptophan and tyrosine residues occurs in this region, less variability between proteins is observed than at 280 nm. There is a need to avoid storing buffers in plastic containers because some plastics leach plasticizers, which absorb at ultraviolet (UV) wavelengths. Detergents can also be troublesome because many absorb UV light. If the buffer or protein solution is cold, the outside of the cuvette may need to be wiped between each reading with a lint-free wiper and the readings should be made quickly after placing the cold solution into the cuvette, because atmospheric moisture may condense on the outside of the cuvette producing an erroneously high reading.
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TL;DR: Aggregate analysis of this type is an important supplement to and often more informative than reems of data difficult to summarize from various techniques, such as high-performance liquid chromatography (HPLC) that separate a large number of individual compounds.
Abstract: Publisher Summary This chapter discusses the analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Analyses of the Folin-Ciocalteu (FC) type are convenient, simple, and require only common equipment and have produced a large body of comparable data. Under proper conditions, the assay is inclusive of monophenols and gives predictable reactions with the types of phenols found in nature. Because different phenols react to different degrees, expression of the results as a single number—such as milligrams per liter gallic acid equivalence—is necessarily arbitrary. Because the reaction is independent, quantitative, and predictable, analysis of a mixture of phenols can be recalculated in terms of any other standard. The assay measures all compounds readily oxidizable under the reaction conditions and its very inclusiveness allows certain substances to also react that are either not phenols or seldom thought of as phenols (e.g., proteins). Judicious use of the assay—with consideration of potential interferences in particular samples and prior study if necessary—can lead to very informative results. Aggregate analysis of this type is an important supplement to and often more informative than reems of data difficult to summarize from various techniques, such as high-performance liquid chromatography (HPLC) that separate a large number of individual compounds .The predictable reaction of components in a mixture makes it possible to determine a single reactant by other means and to calculate its contribution to the total FC phenol content. Relative insensitivity of the FC analysis to many adsorbents and precipitants makes differential assay—before and after several different treatments—informative.
14,046 citations
TL;DR: With the addition of an antioxidant, L-ascorbic acid, the oxidative damage and resultant toxicity of nano-C60 was completely prevented and damage to cell membranes was observed both with chemical assays, and confirmed physically by visualizing membrane permeability with high molecular weight dyes.
665 citations
Cites methods from "Quantitation of protein."
...Total protein was measured in tissue homogenates using the Bradford method [14,15] and lipid peroxidation was normalized to protein content....
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...Total protein was measured in tissue homogenates using the Bradford method [14,15] and total glutathione was normalized to protein content....
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...Total protein was measured in cellular homogenates using the Bradford method [14,15], and DNA content was normalized to protein content....
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TL;DR: These studies will help in designing a rational enzymatic strategy for the synthesis of nanomaterials of different chemical composition, shapes and sizes as well as their separation.
Abstract: Synthesis of silver nanoparticles using α-NADPH-dependent nitrate reductase and phytochelatin in vitro has been demonstrated for the first time. The silver ions were reduced in the presence of nitrate reductase, leading to the formation of a stable silver hydrosol 10–25 nm diam. and stabilized by the capping peptide. The nanoparticles were characterized by X-ray diffraction, transmission electron microscopy, X-ray photoelectron spectroscopy and UV-Vis absorption. These studies will help in designing a rational enzymatic strategy for the synthesis of nanomaterials of different chemical composition, shapes and sizes as well as their separation.
566 citations
TL;DR: This work shows that the MS signal of histones can be used as a “proteomic ruler” because it is proportional to the amount of DNA in the sample, which in turn depends on the number of cells, and adds an absolute scale to the MS readout and allows estimation of the copy numbers of individual proteins per cell.
495 citations
TL;DR: It is proposed that the TPX1 product is involved in the synthesis of lignin and suberin, a tomato peroxidase gene the authors have previously isolated.
Abstract: The last step in the synthesis of lignin and suberin has been proposed to be catalyzed by peroxidases, although other proteins may also be involved. To determine which peroxidases are involved in the synthesis of lignin and suberin, five peroxidases from tomato (Lycopersicon esculentum) roots, representing the majority of the peroxidase activity in this organ, have been partially purified and characterized kinetically. The purified peroxidases with isoelectric point (pI) values of 3.6 and 9.6 showed the highest catalytic efficiency when the substrate used was syringaldazine, an analog of lignin monomer. Using a combination of transgenic expression and antibody recognition, we now show that the peroxidase pI 9.6 is probably encoded by TPX1, a tomato peroxidase gene we have previously isolated. In situ RNA hybridization revealed that TPX1 expression is restricted to cells undergoing synthesis of lignin and suberin. Salt stress has been reported to induce the synthesis of lignin and/or suberin. This stress applied to tomato caused changes in the expression pattern of TPX1 and induced the TPX1 protein. We propose that the TPX1 product is involved in the synthesis of lignin and suberin.
466 citations
Cites methods from "Quantitation of protein."
...Protein was monitored in the chromatographic procedures by their A280 and A260 (Stoscheck, 1990), and in the extracts by the method of Bradford (1976) using bovine serum albumin as the standard....
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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: 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.
225,085 citations
TL;DR: This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique and demonstrates a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts.
20,907 citations
Book•
01 Jan 1963
TL;DR: Methods of enzymatic analysis, Methods of enzymes analysis, the authors, Methods of enzyme analysis, enzymatics, methods of enzymes, and methods of analysis, method of enzymes.
Abstract: Methods of enzymatic analysis , Methods of enzymatic analysis , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی
18,100 citations
TL;DR: The original Lowry method of protein determination has been modified by the addition of sodium dodecyl sulfate in the alkali reagent and an increase in the amount of copper tartrate reagent to be used with membrane and lipoprotein preparations without prior solubilization or lipid extraction.
6,336 citations