The Bradford Method for Protein Quantitation
TL;DR: A rapid and accurate method for the estimation of protein concentration is essential in many fields of protein study, but is susceptible to interference from a wide range of compounds commonly present in biological extracts.
Abstract: A rapid and accurate method for the estimation of protein concentration is essential in many fields of protein study. The Lowry method ( Chapter 1 in vol. 1 of this series) has been widely used, but is susceptible to interference from a wide range of compounds commonly present in biological extracts. Although interference can be avoided by trichloracetic acid precipitation of the protein prior to assay, this lengthens the procedure.
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TL;DR: Hydrophobically modified chitosan containing 5.1 deoxycholic acid groups per 100 anhydroglucose units was synthesized by an EDC-mediated coupling reaction and an efficient of COS-1 cells was achieved by self-aggregates/DNA complexes.
375 citations
01 Jan 2000
TL;DR: In this article, the authors proposed two principles related to the volumetric methods: the direct volume estimation of settled plankton and the measurement of the amount of water displaced by the plankton.
Abstract: Publisher Summary The determination of the total plankton biomass or biovolume is rapid compared with the enumeration and identification techniques. Many samples can be processed in parallel. These measurements are suitable for mixed samples as well as for samples of selected individual species. There is a wide variety of biovolume and biomass measurements. The effort involved in sample treatment increases within the conventional methods and is generally large in biochemical procedures. The methodological bias decreases stepwise: when the amount of excess and interstitial water is reduced by measuring displacement volume or wet mass instead of settled volume, when body fluids are totally removed during dry mass determination, and when the inorganic substances are subtracted. Volumetric methods are the only choice if the samples are to be used for species identification as well. Volumetric methods are also recommended as a quick-look procedure; that is, on board a ship, and in cases where no microbalance or other sophisticated equipment is available. There are two principles related to the volumetric methods: the direct volume estimation of settled plankton and the measurement of the amount of water displaced by the plankton. The first method is more gentle but less precise.
350 citations
TL;DR: The gene that encoded the cytosolic class II smHSP in Arabidopsis thaliana was characterized and its expression was induced by heat and osmotic stress, as well as during seed development, suggesting stress-induced post-transcriptional regulation of At-HSP17.6A expression.
Abstract: Owing to their sessile lifestyle, it is crucial for plants to acquire stress tolerance. The function of heat-shock proteins, including small heat-shock proteins (smHSPs), in stress tolerance is not fully explored. To gain further knowledge about the smHSPs, the gene that encoded the cytosolic class II smHSP in Arabidopsis thaliana (At-HSP17.6A) was characterized. The At-HSP17.6A expression was induced by heat and osmotic stress, as well as during seed development. Accumulation of At-HSP17.6A proteins could be detected with heat and at a late stage of seed development, but not with osmotic stress, suggesting stress-induced post-transcriptional regulation of At-HSP17.6A expression. Overproduction of At-HSP17.6A could increase salt and drought tolerance in Arabidopsis. The chaperone activity of At-HSP17.6A was demonstrated in vitro.
304 citations
TL;DR: The host response to pneumococci was investigated after intranasal inoculation of CD1 mice with 107 log-phase CFU of bacteria, and five major pathogenesis steps from initial infection to death were identified.
Abstract: There is a need for more insight into the pathogenesis of Streptococcus pneumoniae pneumonia, as the fatality rate associated with this disease remains high despite appropriate antibiotherapy. The host response to pneumococci was investigated after intranasal inoculation of CD1 mice with 107 log-phase CFU of bacteria. We identified five major pathogenesis steps from initial infection to death. In step 1 (0 to 4 h), there was ineffective phagocytosis by alveolar macrophages, with concurrent release of tumor necrosis factor alpha (TNF), interleukin-6 (IL-6), and nitric oxide (NO) in bronchoalveolar lavage (BAL) fluid, TNF, IL-6, and interleukin-1 alpha (IL-1) in lung tissues, and IL-6 in serum, which were associated with tachypnea and hemoconcentration. In step 2 (4 to 24 h), bacterial growth in alveoli and polymorphonuclear cell recruitment from bloodstream to lung tissue (high myeloperoxidase levels) to alveoli were associated with high release of all three cytokines and leukotriene B4 (LTB4) in tissue and BAL fluid, as well as transient spillover of IL-1 in serum. In step 3 (24 to 48 h), despite downregulation of TNF and IL-1 in BAL fluid and lungs, there was appearance of injury to alveolar ultrastructure, edema to interstitium, and increase in lung weight as well as regeneration of type II pneumocytes and increased secretion of surfactant; bacteria progressed from alveoli to tissue to blood, and body weight loss occurred. In step 4 (48 to 72 h), strong monocyte recruitment from blood to alveoli was associated with high NO release in tissue and BAL fluid, but there was also noticeable lymphocyte recruitment and leukopenia; bacteremia was associated with TNF and IL-6 release in blood and thrombocytopenia. In step 5 (72 to 96 h), severe airspace disorganization, lipid peroxidation (high malondialdehyde release in BAL fluid), and diffuse tissue damage coincided with high NO levels; there was further increase in lung weight and bacterial growth, loss in body weight, and high mortality rate. Delineation of the sequential steps that contribute to the pathogenesis of pneumococcal pneumonia may generate markers of evolution of disease and lead to better targeted intervention.
239 citations
TL;DR: The results suggest that permanganate is a better choice than ozone for controlling algae derived pollutants and disinfection byproducts after preozonation.
Abstract: Aqueous suspensions of Microcystis aeruginosa were preoxidized with either ozone or permanganate and then subjected to chlorination under conditions simulating drinking water purification. The impacts of the two oxidants on the algal cells and on the subsequent production of dissolved organic matter and disinfection byproducts were investigated. Preozonation dramatically increased disinfection byproduct formation during chlorination, especially the formation of haloaldehydes, haloacetonitriles, and halonitromethanes. Preoxidation with permanganate had much less effect on disinfection byproduct formation. Preozonation destroyed algal cell walls and cell membranes to release intracellular organic matter (IOM), and less than 2.0% integrated cells were left after preozonation with the dosage as low as 0.4 mg/L. Preoxidation with permanganate mainly released organic matter adsorbed on the cells' surface without causing any damage to the cells' integrity, so the increase in byproduct formation was much less. More organic nitrogen and lower molecular weight precursors were produced in a dissolved phase after preozonation than permanganate preoxidation, which contributes to the significant increase of disinfection byproducts after preozonation. The results suggest that permanganate is a better choice than ozone for controlling algae derived pollutants and disinfection byproducts.
216 citations
References
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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
"The Bradford Method for Protein Qua..." refers methods in this paper
...An assay originally described by Bradford ( 1 ) has become the preferred method for quantifying protein in many laboratories....
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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
TL;DR: An assay for proteins in solution that depends on the conversion of Coomassie brilliant blue G250 in dilute acid from a brownish-orange to an intense blue color has high reproducibility and can detect less than 1.0 μg of albumin.
2,803 citations
"The Bradford Method for Protein Qua..." refers background in this paper
...If this presents problems, the linearity of the assay can be improved by plotting the ratio of absorbances at 595 and 465 nm, which corrects for depletion of the free dye ( 12 )....
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TL;DR: The Coomassie brilliant blue G assay for proteins described by Bradford (1976) (Anal Biochem72, 248) was reexamined and it was found that the extinction coefficient of the dye-protein complex solution remained constant over the protein concentration range of 08 to 10 μg/ml of solution.
1,495 citations
TL;DR: Modifications to the Coomassie blue G dye-binding assay for protein are described which remove much of the variation previously observed in the response of this assay to different proteins.
1,362 citations
"The Bradford Method for Protein Qua..." refers background in this paper
...The original Bradford assay shows large variation in response between different proteins ( 5-7 )....
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...3. The assay technique described here is subject to variation in sensitivity between individual proteins (see Table 2). Several modifications have been suggested that reduce this variability ( 5-7 , 9). Generally, these rely on increasing either the dye content or the pH of the solution....
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