Bradford protein assay

About: Bradford protein assay is a research topic. Over the lifetime, 635 publications have been published within this topic receiving 239107 citations.

Sample Preparation for Electrophoresis

Abstract: While standardized protein assays are used in nearly every laboratory, the limitations of many of these assays are frequently overlooked. It is important that an investigator realize that most protein assays are relative and are based on the measurement of particular amino acid residues and/or peptide bonds. The estimation of protein in different assays will therefore depend on the abundance of those amino acids in the protein sample. Because different protein assays may detect different amino acid residues, totally different estimations of protein content may be obtained for the same sample when different methodologies are used. It may therefore be necessary to use more than one protein assay to determine which is optimal for your needs. Table 3.1 summarizes amino acid residues or other chemical bases for protein estimation by different protein assay methods. The commonly used methods for determining total protein have been described elsewhere by Lowry et al. (1951), Warburg and Christian (1941), and Bradford (1976) and have recently been reviewed in detail by Petersen (1983). This latter review also presents a detailed list of chemicals including buffers and detergents that interfere with some of these protein assays.

Određivanje koncentracije proteina-usporedba Bradford, Lowry i Buret metode

Abstract: Determination of protein concentration in a simple is a important step in every analysis. Today different methods for quantitation of protein are developed, but classic spectrophotometric methods, such as Biuret, Lowry i Bradford method, are the most often used in biochemical and medical research. All methods have some advantages and disadvantages, and they are based on reaction of metal ions and dye with proteins to give caracteristically coloured product. Measurment of absorbance are preformed at wavelenght of 540 nm for Biuret method, and at 595 nm for Lowry and Bradford method. The aim of this study is determine and compare sensitivity of Biuret, Lowry i Bradford method fod determination of concentration of proteins, which are isolated from human cervical carcionoma cells, also known as HeLa cells. Concentration of proteins are also determined by modified Bradford method, where for preparation of Bradford reagent, CBB R-250 are used instead of CBB G-250. The highest concentration of proteins are determined by modified Bradford method, but the lowest concentration are determined by use commercial Bradford reagent. This thesis also contains a part with teaching methods on high school level including printed preparation for lesson „Aminoacids and proteins“, corresponding working papers and two experiments for group work. This lesson is prefomed through combination of a lecture and group work (students are divided into 4 groups and they preform two experiments).

The Fusion Expression and Purification of OS-9 Gene and Preparation of Its Polyclonal Antibody

Abstract: OS 9 gene is ubiquitously expressed in human tissues and frequently coamplified with CDK4 gene in human sarcomas, which suggests that the protein may be involved in cell growth or tumor genesis. To obtain soluble protein of OS 9 and its polyclonal antibody for studying the function of OS 9,the fragment of OS 9 gene obtained in the yeast two hybrid system was amplified and cloned into the expression vector pET28a.Induced by IPTG,soluble protein of interest could be obtained. The protein was purified by metal chelate affinity chromatography and its purity and content were detected by thin layer scan analysis and Bradford assay. Rabbits were immunolized by OS 9 protein.The titer and specificity of polyclonal antibody were detected by indirect ELISA assay and Western blotting. Thin layer scanning showed that its purity reached at least 90%.The titer of antiserum was as high as 1∶3200 ,with very high spcificity detected by Western blotting. Through IPTG induction and metal chelate affinity chromatography purification,soluble OS 9 protein with high purity and its specific antibody have been obtained.

Determination of Protein Content in Alhydrogel ® -Based Vaccines by O -Phthalaldehyde Assay

Abstract: The quantification of antigens adsorbed to aluminum-based adjuvants (alum) typically involves a method that first extracts antigen from the alum followed by the quantification of the antigen available in the extract. Extraction procedures often result in less than 100 % desorption of the antigen from the alum adjuvant and may alter the conformation of the antigen, reducing the accuracy of the subsequent method used for quantification. There is no generic method available for directly assessing the protein content when formulated on alum. Here we offer a method that can directly quantify protein adsorbed to Alhydrogel® using a simple fluorescence assay that is highly accurate and reproducible for Alhydrogel® formulations containing 25-400 μg/mL of antigen.

Research update: Lectin enriched fractions of herb and dry extract of Urtica dioica L.

Abstract: Urtica dioica L is a plant rich in flavonoids, carotenoids, caffeoylmalic acid and has an established medical value. Although content of mineral and organic substances of U. dioica L. herb is well characterized, presence of bioactive polypeptides is much less appreciated. Seeds and roots of nettle, have been established as a common source for isolation of lectins. Therefore data on the presence of lectins in herb of nettle is ambiguous. Lectin-enriched protein fractions were isolated from herb (fresh and dry) and dry extract of U. dioica L. by using homogenisation with fluid nitrogen, extraction in 0.01 M phosphate-buffer saline (PBS), concentrating, salting and precipitation. The amount of protein was measured using photometric Bradford method. A proteomic analysis using 2D gel electrophoresis was performed for lectin – enriched protein fractions isolation and analysis. We estimated quantity of protein and lectins, assessed their blood cell agglutinating activity using tests employing rabbit erythrocytes. The highest concentration of protein and specific hemagglutination activity was observed for protein fractions isolated from fresh herb. The highest lectins content was presented in protein fractions isolated from the dry extract. Key words: Urtica dioica L., lectins, hemaglutination, electrophoresis.