Showing papers on "Bradford protein assay published in 2012"

The effects of different preservation processes on the total protein and growth factor content in a new biological product developed from human amniotic membrane

Abstract: The aim of this work is to quantify the total protein and growth factors content in a tissue-suspension obtained from processed human amniotic membrane (hAM). hAM was collected, frozen, freeze dried, powdered and sterilized by γ-irradiation. At each step of the process, samples were characterized for the total protein amounts by a Bradford protein assay and for the growth factor concentrations by ELISA test of the tissue suspensions. Frozen-hAM samples show higher release of total proteins and specific growth factors in the tissue suspension in comparison with freeze-dried hAM. We observed that even if the protein extraction is hindered once the tissue is dried, the powdering process allows a greater release in the tissue suspension of total proteins and growth factors after tissue re-solubilization in comparison with only the freeze-drying process (+91 ± 13% for EGF, +16 ± 4% for HGF, +11 ± 5% for FGF, +16 ± 9% for TGF-β1), and a greater release of EGF (85 ± 10%) in comparison with only the freezing process, because proteins become much readily solubilized in the solution. According with these results, we describe a protocol to obtain a new sterile biological product from hAM tissue, with well-known effects of thermal, mechanical and physical processes on the total protein and grow factors contents.

Uric acid biosensor based on chemiluminescence detection using a nano-micro hybrid matrix

Abstract: Uric acid (UA) is a nitrogenous end product of the purine catabolism in humans. Excessive production of UA may lead to hyperuricemia, gout, and kidney disorders. The study aims at developing an indirect enzymatic UA biosensing assay based on H2O2 detection. The UA biosensing assay was prepared by loading pentacene in peroxalate nanoparticles and Uricase co-immobilized in alginate microspheres. These biosensing formulations were characterized using DLS, optical microscopy, SEM, TEM, and CLSM. Nanoparticles embedded in alginate microspheres formed were used for H2O2 and UA sensing. The dye loaded nanoparticles and co-encapsulated microspheres were formed with size ranges from 520 nm and 60 μm, respectively. Uricase loading using atomization evaluated using Bradford assay was found to be greater than 98% in all tested concentrations. Sensing studies for H2O2 and UA were carried out using fluorescence spectroscopy. H2O2 could be detected in a linear range of 0–6 μM with a regression coefficient of 0.979 and in a range of 6–50 μM with a regression coefficient of 0.984. The limit of detection for each of the range was found to be 1 and 3 μM, respectively. UA sensing showed regression constant 0.977 and 0.949 in range of 0–0.1 mg/ml and 0.015–0.04 mg/ml, respectively. Thus chemiluminescent alginate microspheres containing peroxalate nanoparticles co-immobilized with uricase can be used as diagnostic markers for detection of UA.

Immobilization of Cellulase for Industrial Production

Abstract: Immobilized enzymes are used in analytical chemistry and as catalysts for the production of chemicals, pharmaceuticals and food. Because of their particular structure, immobilized enzymes require optimal conditions, different from those of soluble enzymes. Particle size, particle-size distribution, mechanical and chemical structure, stability and the catalytic activity, used for immobilization, must be considered. Generally, cellulases are used in various industries, including food, brewery and wine, agriculture, textile, detergent, animal feed, pulp and paper, and in research development. For the industrial application of cellulase, its immobilization, which allows the conditions of repeated use of the enzyme alongside retaining its activity, has been recently investigated. Celullase was immobilized with the use of glutaraldehyde, a covalent cross-linking agent in to cross-linked enzyme aggregates (CLEAs). The stability and activity of cross-linked cellulase, exposed to carbon dioxide under high pressure, were studied. Efficiency of enzyme immobilization was determined using Bradford method (Bradford, 1976). The activity of cross-linked cellulase was determined by spectrophotometric method.

Quantitative evaluation of proteins with bicinchoninic acid (BCA): resonance Raman and surface-enhanced resonance Raman scattering-based methods

Abstract: A rapid and highly sensitive bicinchoninic acid (BCA) reagent-based protein quantitation tool was developed using competitive resonance Raman (RR) and surface-enhanced resonance Raman scattering (SERRS) methods. A chelation reaction between BCA and Cu+, which is reduced by protein in an alkaline environment, is exploited to create a BCA–Cu+ complex that has strong RR and SERRS activities. Using these methods, protein concentrations in solutions can be quantitatively measured at concentrations as low as 50 μg mL−1 and 10 pg mL−1. There are many advantages of using RR and SERRS-based assays. These assays exhibit a much wider linear concentration range and provide an additional one (RR method) to four (SERRS method) orders of magnitude increase in detection limits relative to UV-based methods. Protein-to-protein variation is determined using a reference to a standard curve at concentrations of BSA that exhibits excellent recoveries. These novel methods are extremely accurate in detecting total protein concentrations in solution. This improvement in protein detection sensitivity could yield advances in the biological sciences and medical diagnostic field and extend the applications of reagent-based protein assay techniques.

U-2012: An improved Lowry protein assay, insensitive to sample color, offering reagent stability and enhanced sensitivity

Abstract: Traditional colorimetric protein assays such as Biuret, Lowry, and modified Lowry (U-1988) are unsuitable for colored biological samples. Here we describe an improved Lowry protein assay (U-2012), which utilizes stable reagents and offers enhanced sensitivity over the U-1988 assay. U-2012 circumvents interference from colored pigments and other substances (for example sugars) bound to perchloric acid (PCA) precipitated proteins by hydrogen peroxide (H2O2) induced oxidation at 50°C. Unused hydrogen peroxide is neutralized with sodium pyruvate before protein estimation for a stable end color. The U-2012 assay is carried out on the PCA precipitated protein pellet after neutralization (with Na2CO3 plus NaOH), solubilization (in Triton-NaCl), decolorization (by H2O2) and pyruvate treatment. Protein contents in red wine and homogenates of beetroot and blueberry are calculated from standard curves established for various proteins and generated using a rectangular hyperbola with parameters estimated with Microsoft Excel's Solver add-in. The U-2012 protein assay represents an improvement over U-1988 and gives a more accurate estimation of protein content.

A new method of spectrophotometric determination of poly(diallyldimethylammonium chloride) concentration

Abstract: Summary – Spectrophotometric method of protein determination, widely known as the Bradford method, has been applied for quantitative determination of poly(diallyldimethylammonium chloride) (PDDA) monomer units in aqueous solution. In phosphoric acid environment the PDDA polymer stabilizes blue form of the Commasie Briliant Blue G-250 dye having a broad absorption band. Increase in absorbance at wavelengths in the range from 570 to 630 nm follows the increase in monomer units of polymer concentration up to 0.042 mmole/dm 3 . Calibration curves fulfilling second-degree polynomial equations can be determined for PDDA monomer units concentrations not exceeding 0.03 mmole/dm 3 .

Novel peptide-protein assay for identification of antimicrobial peptides by fluorescence quenching.

Abstract: The specific interaction of peptides with proteins is often a key factor which determines biological activities. The determination of K d values of such interactions is commonly performed with fluorescence polarization. However, fluorescence polarization assays are prone to false-positive results due to the potential for non-specific interactions and only afford very low signal-to-background ratios. Here, we present as an alternative a fluorescence resonance energy transfer based quenching assay to measure peptide–protein interactions in solution. In a test setup where antimicrobial peptides were tested for their affinity towards the protein DnaK, the assay provided high specificity and good reproducibility and correlated with the results obtained by fluorescence polarization methods. Furthermore, we established a fast prescreening method which will allow a highly efficient screening of peptide libraries by reducing the amount of sample by 98 % compared to conventional fluorescence polarization assays.

Preliminary study of extractable protein binding using maleic anhydride copolymer

Abstract: A preliminary study of using maleic anhydride copolymer for protein binding has been carried out. The polymeric films were prepared by compression of the purified resin and annealing the film to induce efficient back formation of the anhydride groups. The properties of the film surface were analyzed by attenuated total reflection Fourier transforms infrared spectroscopy and water contact angle measurements. The protein content was determined by Bradford assay. To obtain optimum conditions, immersion time for protein binding was examined. Results revealed that proteins can be successfully immobilized onto the film surface via covalent linkage. The efficiency of the covalent binding of the extractable protein to maleic anhydride-polyethylene film was estimated at 69.87 μg/cm2, although the film had low anhydride content (3%) on the surface.

Adsorption of hemoglobin, fatty acid and glucose to iron nanoparticles as a mean for drug delivery

Abstract: Recent research in biomedicine has documented the application of iron-based nano-particles in supplying the human body with essential nutrients or in drug delivery systems. In this work, the possibility of using iron nanoparticles sorption of biomaterial, such as hemoglobin, cholesterol, triglyceride, serum albumin, and glucose, to cure some specific syndromes have been studies. Bradford assay was used for protein adsorption measurement in supernatant, and FTIR was used for studying the ligands attached to iron nanoparticle. Cholesterol kit and Triglyceride kit were used to measure the cholesterol and triglyceride in supernatant. The result showed that glucose was adsorbed, and the remaining polysaccharide left in supernatant was 42%. Also sorption of triglyceride, cholesterol and serum albumin by nanoparticles was 69%, 73.3% and 87% respectively. FTIR showed hemoglobin being adsorbed to nanoparticles as a ligand. These results indicate that iron nano particles have the capability of being used as a delivery system for biomaterials.

A novel approach for increasing sensitivity in lateral flow assays: Development of an enrichment module based on polyethylene sintered bodies

Abstract: Lateral flow assays (LFAs, eg the well established pregnancy test) are frequently used, fast and easy-to-handle immunoassays with a broad application range Nevertheless, the restriction to small sample volumes is one of the major drawbacks and can be the reason for lack of sensitivity In order to detect even small amounts of analyte in big sample volumes without the need for time consuming sample preparation or sophisticated labelling or detection technologies, the aim of the here presented research was the development of an ‘enrichment module’, which can be integrated into the workflow of a LFA The core element of this enrichment module was a cleavable biological interface structure A fusion protein, which was expressed recombinant and consisted of a monomeric concanavalin A (Con A) domain and a monomeric streptavidin domain (SAv), was used as interface structure For the construction of the entire module, the polysaccharide mannan was firstly covalently attached to a porous polyethylene (PE) sintered body in a four-stage procedure This immobilisation procedure was monitored by a modified Bradford assay In the next step, the Con A–SAv fusion protein was added In order to assess the functionality of the enrichment module, a model assay was developed The SAv domain of the attached fusion protein was specifically recognised by a primary and a secondary, horseradish peroxidase (HRP)-labelled, antibody The HRP-reaction was used for photometric detection in order to characterise binding properties of the enrichment module and to prove binding characteristics of the fusion protein

Determination of Protein in Soybean Stems and Leaves by Bradford Method

Abstract: The content of protein in soybean leaves and stems was determined by Bradford methodThe result showed that it was feasible determining protein content in soybean leaves and stems by this method,as the RSD was 025%～127%;the recovery reached 949%～1064%The average protein content of soybean leaves and stems were 7673% and 11315%,respectivelyThis method was simple,rapid,reproducible,sensitive and accurate,thus was an effective method for determining trace soluble protein

Degradation of earthworm extracts prepared by wet superfine grinding in simulated gastrointestinal environment

Abstract: This is to report the study of degradation of earthworm extracts prepared by wet superfine grinding in simulated gastrointestinal environment. Enzymatic reactions were terminated by adjusting the solution pH or using membrane bioreactor principle. Earthworm protein concentration change was detected by Bradford method, the degraded state of protein was described with SDS-PAGE technology, and the degraded state of small molecule substances was detected by HPLC. The results showed that earthworm protein degraded completely in artificial gastric juice. High molecular weight protein degraded greatly in artificial intestinal fluid, while low molecular weight protein was not significantly degraded. Small molecular substances degradation did not degrade in artificial gastric juice, while they degraded obviously in artificial intestinal fluid, there is even new small molecule substance appeared. Finally it is concluded that the substance that having therapeutic effects in vivo may be some degraded peptide, amino acid and stable small molecules existed in artificial intestinal fluid.

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.

Growth and Protein Profiling by the Cyanobacterium Anacystis nidulans Strains at Different Temperature and Photoperiod

Abstract: Anacystis nidulans was isolated from a shallow Sambhar lake, Jaipur, (Rajasthan), where it occurred in massive amounts. The culture was rendered free from contaminant algae and other cyanobacteria. Anacystis nidulans a prokaryotic, oxygen-evolving, photosynthetic Gram-negative bacteria, survive in a wide variety of extreme environmental conditions [1]. The growth kinetics and physiological properties of phototrophic organisms are markedly influenced by the length of the photoperiod and by the ratio of light to dark hours [2-6]. The importance of the photoperiod has been demonstrated by photosynthesis i.e. a process which delivers energy for carbon assimilation in the light and nitrogen assimilation in the light and in the dark [6-8]. The synthesis of various cellular components is affected by the presence or absence of light-irradiance. In continuous cultures it was found that the total of DNA, RNA and proteins increased at an apparent constant rate during a light-dark cycle [6]. Changes in light regimes and temperatures have been shown to bring about differences in pigment and biochemical composition of microalgae [9-12]. The light/dark photoperiod may be more beneficial than other regimes, as cell number is sustained in exponential phase longer [13]. Experiments suggest that exposure to various light treatments results in a qualitative and quantitative regulation of individual proteins in Synechococcus [14]. Similarly another report showed that the cellular content of nucleic acids and protein decreased during light periods and increased during dark periods in Synechococcus sp. strain PCC 6301 [15].

[Effect of metabolic drugs on the secretion of acylation stimulating protein in 3T3-L1 adipocytes].

Abstract: AIM To develop a 3T3-L1 pre-adipocyte model for evaluating the secretion of acylation stimulating protein (ASP) and an ELISA assay for measuring ASP production and investigate the effects and related potential mechanisms of metabolic drugs on the secretion of ASP, and on the complement C3, triglyceride (TG) mass, nonesterified fatty acids (NEFA) release and fatty acid (FA) uptake into adipocytes METHODS After differentiated, 3T3-L1 pre-adipocytes were treated with chylomicrons, metformin, rosiglitazone, rimonabant for 48 h ASP and C3 were measured using a sandwich ELISA NEFA levels were measured using enzymatic colorimetric kits FA uptake was measured in a bottom-reading fluorescent microplate reader TG mass and protein levels were determined using enzymatic colorimetric assay and the Bradford assay, respectively RESULTS Chylomicrons increased ASP production (up to 411%±133%, P<005) Rosiglitazone and rimonabant decreased ASP production (-53% to -85%, P<005), associated with a decrease in the precursor protein C3 (-37% to -65%, P<001) By contrast, metformin also decreased ASP (-54% to -100%, P<005), but with no change in precursor protein C3 In addition, metformin decreased TG mass (maximum -60%, P<005) and real-time FA uptake (maximum -75%, P<005) CONCLUSION 3T3-L1 pre-adipocyte model is successfully developed, which can be used for evaluating the effects of metabolic drugs on the secretion of ASP ELISA assay for measuring ASP production is also developed

The Optimization of Synthesis Condition of ZnSe Nanocrystals

Abstract: In biomineralization, self-organization of organic based templates provides scaffolding for the assembly of QDs materials. The host-guest relationship between these protein cages and the encapsulated material is based primarily on a complementary electrostatic interaction. Zinc selenide (ZnSe) were synthesized in the cavity of the apoferritin from horse spleen (HsAFr) and the reaction condition was optimized by adding tween 20 to avoid ferritin agglomeration. The obtained nanodots were characterized by TEM, and absorption measurements. In addition, the protein concentration of ZnSe-ferritin was precisely measured by the Bradford protein assay method. From the results, it was concluded that the ZnSe nanocrystals were successfully synthesized in the core of ferritin and it can be applied as a potential functional material such as transistors, biosensor materials or medical imaging.

Ponceau 4R: A Novel Staining Agent for Resolve Food Proteins on PAGE and Its Impact on Digestibility

Abstract: Ponceaue 4R interaction with protein, Nisin and BSA was concentration dependent and may be used for protein assay. As the dye binds with almost all the proteins and current methodology may be used for the estimation of proteins in various food systems. During the course of present work staining with ponceau 4R of resolved proteins on PAGE (poly acryl amide gel electrophorosis) was comparable with Coommassie Brilliant Blue R250. The Ponceaue 4R was highly sensitive, rapid and produced sharp red bands on the gel on 0.2% concentration. The effects of pH, concentration of proteins and dye were also investigated in various conditions which would help food processors to use a calculated amount of dye. The impact of tryptic digestibility on Ponceaue 4R -Protein Complexes (PPC) has illustrated that dye may safely be used without any adverse effect on the digestion of PPC.