Showing papers on "Bradford protein assay published in 2006"

Glomalin-related soil protein: Assessment of current detection and quantification tools

Abstract: Despite the widely acknowledged importance of arbuscular mycorrhizal fungi (AMF) in soil ecology, quantifying their biomass and presence in field soils is hindered by tedious techniques. Hence biochemical markers may be useful, among which glomalin-related soil protein (GRSP) could show a particular promise. Presently GRSP is operationally defined, its identification resting solely on the methods used to extract it from soil (citric acid buffer and autoclaving) and the assays (Bradford/enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody) utilized to detect it. The current assumption is that most non-heat stable soil proteins except glomalin are destroyed during the harsh extraction procedure. However, this critical assumption has not been tested. The purpose of this research was to challenge the GRSP extraction process to determine the accuracy of the Bradford method as a measure of glomalin; and to provide some assessment of the specificity of the ELISA monoclonal antibody. In two studies we spiked soil samples either with known quantities of a glycoprotein (BSA: bovine serum albumin) or with leaf litter from specific sources. After extraction 41–84% of the added BSA was detected with the Bradford method. This suggests that the currently used extraction procedure does not eliminate all non-glomalin proteins. Also, ELISA cross-reactivity against BSA was limited, ranging from 3% to 14%. Additions of leaf litter also significantly influenced GRSP extraction and quantification suggesting that plant-derived proteins, as would occur in the field, had a similar effect as BSA. Litter additions decreased the immunoreactive protein values, suggesting interference with antibody recognition. We conclude that the use of GRSP, especially Bradford-based detection, in the assessment of AMF-derived substances within field soils is problematic, it may be inappropriate in situations of significant organic matter additions.

Quantification of surface-bound proteins by fluorometric assay: Comparison with quartz crystal microbalance and amido black assay.

Abstract: Protein adsorption is of major and widespread interest, being useful in the fundamental understanding of biological processes at interfaces through to the development of new materials. A number of techniques are commonly used to study protein adhesion, but few are directly quantitative. Here we describe the use of Nano Orange, a fluorometric assay, to quantitatively assess the adsorption of bovine fibrinogen and albumin onto model hydrophilic (OH terminated) and hydrophobic (CH3 terminated) surfaces. Results obtained using this method allowed the calibration of previously unquantifiable data obtained on the same surfaces using quartz crystal microbalance measurements and an amido black protein assay. Both proteins were found to adsorb with higher affinity but with lower saturation levels onto hydrophobic surfaces. All three analytical techniques showed similar trends in binding strength and relative amounts adsorbed over a range of protein concentrations, although the fluorometric analysis was the only me...

Monitoring cyclodextrin-polyviologen pseudopolyrotaxanes with the Bradford assay.

Abstract: Self-assembled multivalent pseudopolyrotaxanes, composed of lactoside-bearing cyclodextrin (CD) rings threaded on linear polyviologen polymers, have been introduced recently as flexible and dynamic neoglycoconjugates In the course of this research, it was found that polyviologens are responsive to the Bradford assay, which is traditionally highly selective for proteins The response of the pseudopolyrotaxanes to the Bradford assay was dependant on, and thus indicative of, the degree of threading of the CD rings onto the polyelectrolyte The assay was then used to report on the threading and dethreading of native and lactoside-bearing α-CD rings onto and off of polyviologen chains, a phenomenon which demonstrates the utility of biochemical assays to address problems unique to supramolecular chemistry

Degradation of Acid Red 27 by the Recombinant Flavin Reductase

Abstract: This paper focuses on the degradation of azo compound C.I. Acid Red 27 (AR27, amaranth) by the recombinant enzyme flavin reductase (FRE) from Citrobacter freundii strain A1. The enzyme was obtained via re-transformation of recombinant plasmid pET-43.1c(+)freBP containing the flavin reductase gene (fre) into E. coli NovaBlue. The plasmid was subsequently transformed into E. coli BL21(DE3)pLysS for overexpression of FRE fusion protein. Prior to that, the stability of fre gene was first verified using PCR amplification and also sequencing of the nucleotides and consequently compared with the original fre gene sequence from C. freundii A1. The protein was expressed as inclusion bodies and was isolated and refolded in order to obtain a properlyfolded and active flavin reductase. The activity and the protein yield were monitored using a modified flavin reductase assay and Bradford assay respectively. The active enzyme was further subjected to the degradation of AR27 and the degradation profile was constructed.

Purification of COPI Vesicles

Abstract: Publisher Summary This chapter describes the in vitro assay that was developed for the faithful formation, isolation, and characterization of COPI-derived vesicles from highly purified rat liver Golgi membranes. Although the exact mechanism for vesicle formation and cargo incorporation is still debated, the fundamental coat components, as well as the basic principles of vesicle formation, have been clear for some time. The hydrolysis of GTP by ADP-ribosylation factor 1 (Arfl), stimulated by a GTPase-activating protein (GAP), is then thought to trigger vesicle uncoating, producing a transport intermediate capable of fusing with target membranes. After terminating the reaction, slowly sedimenting vesicles are separated from heavier donor membranes by medium speed centrifugation. The COPI-derived vesicles are then recovered using high speed centrifugation onto a two step sucrose cushion. Protein concentration determination is performed using the Bio-Rad version of the Bradford protein assay reagent and BSA as a standard. Pipetting of Golgi membranes should be minimized to avoid shearing. Mix these aliquots by tapping carefully on the side of the tube.

Determination of Concentration of Laccase by Atomic Absorption Spectrometry

Abstract: The concentration of copper in laccase solution was determined by atomic absorption spectrometry,and the standard curve of the concentration of copper to the concentration of laccase was linear within 0~1 mg·mL~(-1)(R= 0.9931).The immobilization amount and the immobilization rate of laccase determined by atomic absorption spectrometry were slightly lower than those by Bradford method when BSA was used as reference protein.Results showed atomic absorption spectrometry was an easy way to determine the concentration of laccase.

Chapter 17 – Protein Determination

Abstract: Publisher Summary This chapter focuses on three techniques that can be used to determine the protein content of tissues or samples. The three techniques include Lowry Assay, Bradford Assay, and Neuhoff Assay. All assays described in this article quantitate protein relative to a standard protein. The choice of the standard protein can markedly influence the result. This requires special attention for proteins with a high content of certain amino acids. The most efficient way to prepare an exact dilution series of the standard protein employs a handheld dispenser. Many reagents used commonly in protein extraction interfere with Lowry assay. The dot-blot assay is a versatile tool. Its different modifications enable one to cope with almost every potentially interfering substance. The dot-blot assay combines high sensitivity, an extended range of linearity, and high tolerance to potentially interfering substances. The Lowry assay exhibits the best accuracy with regard to absolute protein concentrations due to the chemical reaction with polypeptides. It is also useful for the quantitation of oligopeptides.