Showing papers on "Bradford protein assay published in 2001"

Agent for protein precipitation, a method of protein precipitation, a method of protein assay using protein precipitation agent, and a kit for protein assay

Abstract: A method of protein precipitation, concentration and removal of non-protein agents from the protein solution wherein the protein solution is treated with a protein-precipitation agent containing an acidic agent, a salt and a precipitate forming agent. After precipitation, the protein precipitate is washed with a water miscible organic solvent agent to remove non-protein agents present in the protein precipitate.

Assays for Total Protein

Abstract: This unit describes three copper-based assays to quantitate total protein: the biuret method, a variation of the Lowry method (Hartree-Lowry method), and the bicinchoninic acid (BCA) assay. Acid hydrolysis of a protein is coupled with ninhydrin detection to quantitate amino acid content of a sample. Ultraviolet spectrophotometry is used to measure total protein and evaluate samples for the presence of contaminants. The Coomassie dye binding, or Bradford, assay is a quite simple assay and frequently is quite sensitive, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acid pH. Finally, dry weight measurement is used to quantitate pure protein. Support protocols describe heat sealing glass tubes for acid hydrolysis, sample dialysis in polyacrylamide gel wells to remove low-molecular-weight contaminants, and TCA precipitation to precipitate and concentrate proteins and remove low-molecular-weight contaminants.

17 - Expression, Purification, and Biochemical Properties of Rabkinesin-6 Domains and Their Interactions with Rab6A

Abstract: This chapter describes the purification of several Rabkinesin-6 domains from bacteria and eukaryotic systems, and their biochemical characterization (ATPase, MT-binding, hydrodynamic properties)The chapter describes an assay to visualize the interaction between Rab6A and Rabkinesin-6 For purification, Escherichia coli strain freshly transformed with pET-Nt or pTrc-Q665 plasmids are cultured Bacterial pellets are obtained are sonicated, centrifuged, and chromatographed Protein fractions are analyzed by SDS-PAGE and Western blot This protocol leads to the purification of about 05–1 mg fusion proteins α/β-Tubulin is purified from bovine brain with two cycles of polymerization in the presence of GTP, and microtubule (MT)-associated proteins (MAPs) are removed by phosphocellulose chromatography Tubulin concentration is estimated by the Bradford assay and MT concentration is expressed per tubulin heterodimer MT-induced ATPase activity of kinesins is often measured by using a NADH coupled enzymatic assay

Evaluation of desorption of proteins adsorbed to hydrophilic surfaces by two-dimensional electrophoresis.

Abstract: The evaluation of the plasma protein adsorption patterns of superparamagnetic iron oxide (SPIO) particles is of high interest concerning their in vivo fate and is carried out by two-dimensional electrophoresis (2-DE). The sample preparation is of great importance, especially the removal of the adsorbed proteins (desorption) from the particle surface for subsequent analysis by 2-DE. The removal is carried out by a desorption solution. In this study, negatively and positively charged SPIO model particles were under investigation concerning the desorption of proteins adsorbed on their surfaces. Firstly, the desorption process was determined quantitatively using the Bradford protein assay. Secondly, the removable or nonremovable protein species, from particles surface were under investigation by 2-DE. Looking at the desorption in a quantitative manner with the Bradford assay, the desorption efficacy from negatively charged particles was about 90%. In the case of the positively charged particles, the desorption efficacy seemed to be reduced, approximately 34% of the proteins remained on the surface. Comparing the protein patterns of the particles evaluated by 2-DE in the desorption solution and the proteins remaining on the particles, they confirmed the results from the protein quantification. After desorption, the IgG gamma-chains were found to be the dominant protein fraction remaining on the negatively charged particles. On the positively charged particles, many more protein species were found after desorption. The more basic the protein fragments, the more ineffective was the desorption from the positively charged model particle, and vice versa. Nevertheless, all protein spots were found qualitatively in the desorption solution, especially when the desorption solutions still containing the particles were used for the 2-DE analysis. In conclusion, 2-DE could be confirmed as the "gold standard" for determining the plasma protein adsorption patterns of nanoparticulate systems.

Protein quantification with coomassie bulliant blue microplate-colormetric

Abstract: AIM To develop a rapid and sensitive method of protein quantification assay. METHODS Protein content of bovine serum albumin (BSA) and mice liver homogenate was determinated by microplate colormetric (MPC) in which coomassie brilliant blue (CBB) is used as color reagent. RESULTS Detecting limit of MPC was lower (0.63 μg) than that of macro colormetric (MC). MPC had advantage of rapidity and lesser volume of sample (a few microliter). CONCLUSION MPC is a method of simplicity, rapidity and sensitivity for protein quantification assay especially suitable for the determination of large number of microliter samples.

High-sensitive trace protein assay by argentation

Abstract: PROBLEM TO BE SOLVED: To provide an assay capable of high-sensitively and easily determining trace amounts of urinary protein in large numbers of specimens without using an immunological method. SOLUTION: A silver colloid is formed in a stain solution by preliminarily reacting a silver ion with a reducing agent comprised of ferrous sulfate. A protein in a cellulose acetate film can be determined using a method for combining the protein with the silver colloid to stabilize it. A certain amount of the specimen is spotted directly on the cellulose acetate film in a dry state without buffering. The protein in this state is fixed by sulfosalicylic acid and trichloroacetic acid, and then well cleaned by a diluted acetic acid solution. After the cleaning, the protein is stained with the argentation solution.