Bovine serum albumin , protein precipitation assay

In various runs, specific activity went from an average of 2.75 lU/mg protein (based on the Bio-Rad protein assay with bovine serum albumin standard) for Ae centrifuged crude preparation after alcohol precipitation, to 8 lU/mg after DEAE-cellulose chromatography, to 100 lU/mg after DEAE-Fractogel chromatography, to 1000 RJ/mg after Sephadex G-50 chromatography, and to 3200 lU/mg after CM-Trisacryl chromatography. 

Biophysical characterization showed that a single HRP II protein bound 17 molecules of heme [35]. In an in vitro heme polymerization assay, HRP II promoted the synthesis of hemozoin, while controls, such as the proteins bovine serum albumin and lysozyme or the homopeptides polyhistidine, polylysine, and polyasparagine, did not. FT-IR analysis of the reaction product showed the characteristic vibrations of hemozoin. The polymerization activity had a pH maximum near 4.0, which dropped off precipitously near the pKa of histidine. The heme polymerization... 

The assay mixture consisted of 1.3 mg/mL microsomal proteins, 50 mM phosphate buffer (pH 8.0), 10 mM MgCl2, and 10 pM thyroxine (completely solubilized in 1% bovine serum albumin-NaOH) containing 0.5 pCi/nmol [125I]thyroxine. The reaction was initiated by the addition of 2 mM UDP-glucuronic acid. After incubation at 37°C, the reaction was stopped by addition of an equal volume of methanol-orthophosphoric acid (9 1, v/v). Precipitated proteins were removed by centrifugation before injection of an aliquot into the HPLC system. Reactions were linear for at least 60 minutes. 

Precipitation reactions of polyphenols with proteins depend on the concentration and structure of the compounds and proteins involved, the pH in the reaction mixture as well as on the presence of modifiers (genuine in the extracts or reagents) [47-48]. Studies on the precipitation reaction in solution showed that the affinities of proteins to polyphenols varies over several orders of magnitude. High affinity was found for conformationally loose, proline-rich proteins which are strong hydrogen bond acceptors [156]. Bovine serum albumine (BSA) is currently the most often used protein for precipitation assays. 

Protein Binding—Tannins, as the name implies, characteristically form complexes with proteins based on multipoint hydrogen bonding, and this feature can be used to detect and remove tannins from crude extracts. A visible protein-tannin precipitate should be formed on addition of the tannin-containing extract to a relatively concentrated (approx 5%) solution of bovine serum albumin (BSA). The amount of BSA either remaining in solution or in the precipitate can be analyzed by various protein determination assays. 

Due to the high cost of cell culture, Caco-2 assays are usually used as a follow-up to PAMPA in ADME screening [78], and as a result, the sample burden for bioanalysis is not as heavy as for some first-hne assays, such as metabolic stability. There have been a number of reports in the literature that use automated optimization and single LC-MS/MS for sample analysis for Caco-2 assay support [46,79-81]. Nevertheless, Caco-2 samples pose a unique bioanalytical challenge. Unlike plasma or microsomal samples rich in proteins that help solubilize compounds and prevent adsorptive loss, Caco-2 samples are essentially aqueous buffer samples with very little protein. As a result, compounds with low solubility and/ or adsorption problems tend to exhibit poor recoveries in the assay due to precipitation and adsorptive losses [82,83]. An effective solution to this problem is the use of organic solvent to catch compounds immediately after incubation, but prior to analysis, in order to maintain solubility and prevent adsorptive loss to container surfaces. Another approach involves the addition of some protein such as bovine serum albumin (BSA) to the assay buffer system, thus reducing compound loss/ precipitation and improving recoveries [84]. 

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