Showing papers on "Bradford protein assay published in 1996"
TL;DR: Under standard assay conditions, the ratio of the absorbances, 590 nm over 450 nm, is strictly linear with protein concentration, and a linear equation that perfectly fits the experimental data is provided on the basis of mass action and Beer's law.
1,003 citations
TL;DR: It was found that sodium chloride concentration and acidity of the solutions have significant effect on the sensitivity of BPB protein assay, and the Sandell index was used to express theensitivity of protein detection.
79 citations
TL;DR: When gel filtration fractions from the extract of the marine invertebrate Cucumaria echinata were subjected to this assay, different carbohydrate-binding activities were observed with different elution profiles, suggesting that this assay could also be widely applicable for the simultaneous detection of lectins from various sources.
51 citations
TL;DR: A method that involves solubilization of cells with sodium dodecyl sulfate, precipitation of proteins with trichloroacetic acid (TCA) with special physical exclusion of DNA aggregate and reconstitution of precipitated proteins with Tris base is developed.
Abstract: Experimentation with cultured cells often requires analyzing cellular protein extract by gel electrophoresis and immunoblotting. Traditional methods for extracting cellular proteins by homogenization or detergent solubilization usually produce protein samples that are viscous (due to the presence of DNA) and prone to degradation due to the presence of endogenous protease activity. We have developed a method that involves solubilization of cells with sodium dodecyl sulfate (SDS), precipitation of proteins with trichloroacetic acid (TCA) with special physical exclusion of DNA aggregate and reconstitution of precipitated proteins with Tris base. Protein samples prepared by this method contain little DNA, making them ideal for long-term storage. The solubilized total protein extracts are fully compatible with protein assay, gel electrophoresis and Western blotting. When compared to protein extracts from a homogenization method, those from the TCA method showed an identical total protein staining pattern on SDS polyacrylamide gel electrophoresis and contained distinct cellular proteins recognized by many monoclonal and polyclonal antibodies tested (including anti-actin, spectrin, protein kinase C (alpha), talin and spectrin) on Western blots.
45 citations
TL;DR: The multistep assay of specific catechol-O-methyltransferase (COMT) activity in human erythrocytes was validated and demonstrates that the assay was able to detect differences between the subjects and the effect of COMT inhibition in the clinical study.
25 citations
TL;DR: A method for estimating number of dye-binding sites and binding constants from standard curve-fitting procedures is established, and the possibility of protonated forms of the dye binding to proteins was not ruled out by this study.
23 citations
TL;DR: In this article, the enzymic availability of β-lactoglobulin was determined in view of its pH-dependent molecular association status and was best at pH > 7.5, favouring the monomeric structure.
15 citations
TL;DR: Quantitation of antibody coupled to a derivatized polystyrene bead through a bifunctional cross linker can be accomplished by a competitive enzyme-linked immunosorbent assay (ELISA) method.
Abstract: Quantitation of antibody coupled to a derivatized polystyrene bead through a bifunctional cross linker can be accomplished by a competitive enzyme-linked immunosorbent assay (ELISA) method. This sensitive method is less subject to interference than other protein assay methods such as bicinchoninic acid (BCA) or Lowry. The competitive ELISA method consists of incubating the coupled bead with a (20/80) weight ratio of goat anti mouse kappa alkaline phosphatase/goat anti mouse kappa (GAMKAP/GAMK) for 1.5 hours at 37°C, washing, adding p-nitrophenyl phosphate (PNPP) substrate, and reading the absorbance at 405/450 nm. A standard curve is established with radiolabeled antibody beads for microgram quantitation.
12 citations
TL;DR: Interference in the protein assay can be overcome by including H2O2 at a final concentration of 0.1% in theprotein assay medium prior to the addition of the dye reagent, which results in accurate measurements in the tissue preparation containing vanadyl ribonucleoside or orthovanadate.
8 citations
01 Jan 1996
TL;DR: This chapter focuses on two assays for protein: the Lowry assay and the Bradford assay, which are a dye-binding assay based on the differential color change of a dye in response to various concentrations of protein.
Abstract: A variety of procedures have been developed in recent years to quantitate protein in biological specimens. Methodologies continue to be improved and new assays have been developed, which has now made it difficult to decide which assay(s) to use for a sample. The choice, however, is often based on individual idiosyncrasies of laboratory experience or bias in a particular subfield of biology. This chapter focuses on two assays for protein: the Lowry assay and the Bradford assay. The relative merits and limitations of these assays can be evaluated by comparing and contrasting these procedures. The Lowry reaction for protein determination is an extension of the Biuret procedure, but it is more sensitive than the Biuret method and is more time consuming as well. The main precaution to be observed when performing this assay concerns the addition of the Folin reagent. The Bradford protein assay is a dye-binding assay based on the differential color change of a dye in response to various concentrations of protein. The Bradford protein assay is more widely used as compared to the Lowry method as it is comparatively easier to use, requires one reagent, and takes just five minutes to be performed.
1 citations
01 Jan 1996
TL;DR: The glass beads grinding procedure is currently used as the most effective technique for yeast cell extraction as the very thick and tough cell wall of yeast renders methods—such as sonication, osmotic lysis, and homogenization—ineffective in breaking the cell.
Abstract: A variety of methods like sonication, homogenization with a glass or Teflon pestle, grinding with aluminum or glass beads, and high pressurization and rapid release of pressure are used for cell-extract preparation as the extraction of cellular material from whole cells is a critical step in enzyme purification. The preparation of cell extracts (also called cell-free extracts) requires a very careful execution so as to ensure complete release of the enzyme from cellular material without denaturation of the enzyme itself. In case of yeast, the easiest and most effective method for cell-extract preparation involves grinding with glass beads. The shearing action produced by the glass beads during vortexing causes the rupture of the yeast cell. The glass beads grinding procedure is currently used as the most effective technique for yeast cell extraction as the very thick and tough cell wall of yeast renders methods—such as sonication, osmotic lysis, and homogenization—ineffective in breaking the cell. The Bradford protein assay technique has been further elaborated in this chapter.