Showing papers on "Bradford protein assay published in 2008"

Glomalin extraction and measurement

Abstract: We investigated extraction from soil of glomalin, a glycoprotein produced by arbuscular mycorrhizal fungi, and we examined its measurement. The most commonly used protocols for extracting glomalin require autoclaving of soil in citrate solution, followed by centrifugation to separate the supernatant, and then measurement by either Bradford protein assay or enzyme-linked immunosorbent assay (ELISA). We found that lengthening the time of autoclaving increased easily extractable glomalin extraction. Delay of centrifugation after autoclaving, however, diminished Bradford-reactive substances in the supernatant, suggesting that extracted substances might be reversibly immobilized on soil particles. Surprisingly, increasing the volume of extraction solution did not accelerate extraction of “total glomalin”, but instead, substantially increased the amount extracted. Multiple autoclave cycles nevertheless denature glomalin, which may not be as heat-resistant as thought. Repeated 1-h autoclaving of supernatant diminished both its Bradford-reactive substances (7.3% h−1) and immunoreactive protein (22% during the first hour and 9.5% h−1 of the remainder thereafter), although a large initial volume of extractant could reduce the loss of immunoreactive protein. Proteins and polyphenols that survive the extraction process are measured non-specifically by the Bradford assay. When we added other glycoproteins to dry soils, we recovered a maximum 34% bovine serum albumin and 22% bovine mucin, primarily in the first two, 1-h extraction cycles. These added proteins may adhere to soil organic matter and thereby be protected from denaturation. In addressing the endpoint of glomalin extraction, we found that the Michaelis–Menten equation closely fits cumulative glomalin per extraction cycle such that its asymptote provides an objective estimate of total extractable glomalin for a given set of extraction conditions. Additionally, the equation provides a curvature parameter that reflects the soil-specific efficiency of an extraction protocol. Although the soils that we investigated with 7.6% or more soil organic matter had the most asymptotic total glomalin, they were extracted the least efficiently.

Dark-to-bright optical responses of liquid crystals supported on solid surfaces decorated with proteins.

Abstract: Protein assays are critical analytical tools performed in various biochemical laboratories to quantify the concentration of proteins. In this study, we report the optical responses of a thin layer of liquid crystals supported on glass slides decorated with proteins and the utility of this phenomenon as a new “all-or-nothing” type of protein assay. It was found that the orientations of liquid crystals are very sensitive to the concentration of protein solution applied to the surface. When the protein concentration exceeds a critical value (IgG 5.0 μg/mL, BSA 6.0 μg/mL, FTIC-anti-biotin 0.40 μg/mL, and FITC-anti-IgG 0.37 μg/mL), the thin layer of liquid crystals gives a very sharp dark-to-bright optical response within a small concentration range. This characteristic is not observed in any traditional protein assays, which are based on the adsorption of UV or visible light. The optical response is also very precise and reproducible. It is not affected by the thickness of the liquid crystal cell or the amoun...

Quantification of the activity of biomolecules in microarrays obtained by direct laser transfer.

Abstract: The direct-writing technique laser-induced forward transfer has been employed for the micro-array printing of liquid solutions of the enzyme horseradish peroxidase and the protein Titin on nitrocellulose solid surfaces. The effect of two UV laser pulse lengths, femtosecond and nanosecond has been studied in relation with maintaining the activity of the transferred biomolecules. The quantification of the active biomolecules after transfer has been carried out using Bradford assay, quantitative colorimetric enzymatic assay and fluorescence techniques. Spectrophotometric measurements of the HRP and the Titin activity as well as chromatogenic and fluorescence assay studies have revealed a connection between the properties of the deposited, biologically active biomolecules, the experimental conditions and the target composition. The bioassays have shown that up to 78% of the biomolecules remained active after femtosecond laser transfer, while this value reduced to 54% after nanosecond laser transfer. The addition of glycerol in a percentage up to 70% in the solution to be transferred has contributed to the stabilization of the micro-array patterns and the increase of their resolution.

Comparison and Validation of Methods To Quantify Cry1Ab Toxin from Bacillus thuringiensis for Standardization of Insect Bioassays

Abstract: Standardization of toxin preparations derived from Bacillus thuringiensis (Berliner) used in laboratory bioassays is critical for accurately assessing possible changes in the susceptibility of field populations of target pests. Different methods were evaluated to quantify Cry1Ab, the toxin expressed by 80% of the commercially available transgenic maize that targets the European corn borer, Ostrinia nubilalis (Hubner). We compared three methods of quantification on three different toxin preparations from independent sources: enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (SDS-PAGE/densitometry), and the Bradford assay for total protein. The results were compared to those obtained by immunoblot analysis and with the results of toxin bioassays against susceptible laboratory colonies of O. nubilalis. The Bradford method resulted in statistically higher estimates than either ELISA or SDS-PAGE/densitometry but also provided the lowest coefficients of variation (CVs) for estimates of the Cry1Ab concentration (from 2.4 to 5.4%). The CV of estimates obtained by ELISA ranged from 12.8 to 26.5%, whereas the CV of estimates obtained by SDS-PAGE/densitometry ranged from 0.2 to 15.4%. We standardized toxin concentration by using SDS-PAGE/densitometry, which is the only method specific for the 65-kDa Cry1Ab protein and is not confounded by impurities detected by ELISA and Bradford assay for total protein. Bioassays with standardized Cry1Ab preparations based on SDS-PAGE/densitometry showed no significant differences in LC50 values, although there were significant differences in growth inhibition for two of the three Cry1Ab preparations. However, the variation in larval weight caused by toxin source was only 4% of the total variation, and we conclude that standardization of Cry1Ab production and quantification by SDS-PAGE/densitometry may improve data consistency in monitoring efforts to identify changes in insect susceptibility to Cry1Ab.

Tannic acid reduces recovery of water-soluble carbon and nitrogen from soil and affects the composition of Bradford-reactive soil protein

Abstract: Tannins are plant-derived polyphenolic compounds that precipitate proteins, bind to metals and complex with other compounds. Solutions of tannic acid, or other phenolic compounds, were added to soil samples to determine if they would affect recovery of soluble soil carbon (WSC) or –nitrogen (WSN) or influence the extraction and composition of Bradford-reactive soil protein (BRSP), associated with glomalin. Tannic acid-C added with water was not completely recovered from samples and the amount of total net WSC and WSN recovered was reduced, suggesting formation of insoluble complexes. By comparison, non-tannin phenolics like gallic acid, or methyl gallate, had little effect on extraction of WSC or WSN while a simple gallotannin derived from tannic acid, 1,2,3,4,6-penta- O -galloyl- d -glucose (PGG), inhibited extraction most. The C and N concentrations in BRSP increased when soil samples were treated with tannic acid or PGG before extraction, a procedure that includes autoclaving. Increases were greatest in the 10–20 cm compared to 0–5 cm depth. Accompanying these were declines in the ratio of absorbance at 465 and 665 nm ( E 4/ E 6 ratio) of BRSP extracts suggesting formation of larger or heavier molecules. In contrast, C and N composition in lyophilized BRSP was unaffected or even slightly reduced when tannic acid or PGG were added to the BRSP extract solution after the extraction process. We conclude that some tannins can reduce the solubility of labile soil C and N, at least temporarily and given unpredictability of response associated with phenolic substances, the Bradford assay should not be relied on to quantify pools or composition of soil proteins like glomalin.

Evaluating the compatibility of three colorimetric protein assays for two-dimensional electrophoresis experiments.

Abstract: To evaluate compatibility of commonly used colorimetric protein assays for 2-DE experiments, we investigated the interfering mechanisms of major 2-DE component(s) in the Lowry-based assay, the Bradford assay and the bicinchoninic acid (BCA) assay. It was found that some 2-DE components did not directly interfere with the assays' color development reaction, but possibly influenced the quantitation results by interacting with proteins. Generally, simultaneous presence of 2-DE components in the samples demonstrated a cooperative rather than additive interference. Interference by reductants in the Lowry-based assay and the BCA assay were too prominent and could not be completely eliminated by either the reported alkylation procedure or the water dilution procedure. The Bradford assay however, presented a more suitable method for quantitating 2-DE samples because it was less interfered by most 2-DE components. Furthermore, despite slightly compromising protein solubility, utilization of reductant free 2-DE sample buffers conferred application of the Lowry-based and BCA assays in the 2-DE experiments.

Results and reliability of protein quantification for two-dimensional gel electrophoresis strongly depend on the type of protein sample and the method employed.

Abstract: We investigated the effects of tissue samples taken from rat brain on the reliability of three protein quantification kits: the Bradford assay, the 2-D Quant Kit, and the EZQ Protein Quantitation Kit. All three assays measured significantly smaller amounts of protein after extraction than the reference values before extraction. Only small effects were seen in homogenates, but very pronounced differences in membrane-enriched and highly lipophilic subcellular fractions. Researchers should evaluate which method of protein quantification is best qualified for their specific experimental design.

Quantitation of Protein in Samples Prepared for 2-D Electrophoresis

Abstract: The concentration of protein in a sample prepared for two dimensional (2-D) electrophoretic analysis is usually determined by protein assay. Reasons for this include the following. (1) Protein quantitation ensures that the amount of protein to be separated is appropriate for the gel size and visualization method. (2) Protein quantitation facilitates comparison among similar samples, as image-based analysis is simplified when equivalent quantities of proteins have been loaded on the gels to be compared. (3) Quantitation is necessary in cases where the protein sample is labeled with dye before separation (1,2). The labeling chemistry is affected by the dye to protein ratio so it is essential to know the protein concentration before setting up the labeling reaction.A primary consideration with quantitating protein in samples prepared for 2-D electrophoresis is interference by nonprotein substances that may be present in the sample. These samples generally contain chaotropic solubilizing agents, detergents, reductants, buffers or carrier ampholytes, all of which potentially interfere with protein quantitation. The most commonly used protein assays in proteomics research are colorimetric assays in which the presence of protein causes a color change that can be measured spectrophotometrically (3). All protein assays utilize standards, a dilution series of a known concentration of a known protein, to create a standard curve. Two methods will be considered that circumvent some of the problems associated with interfering substances and are well suited for samples prepared for 2-D electrophoresis. The first method (4.1.1) relies on a color change that occurs upon binding of a dye to protein and the second (4.1.2) relies on binding and reduction of cupric ion (Cu2+) ion to cuprous ion (Cu+) by proteins.

A solid-phase assay for the quantitation of total protein eluted from balafilcon, lotrafilcon, and etafilcon contact lenses

Abstract: Purpose: To compare two variations of a membrane-based protein assay utilizing Amido black (AB) detection with a commercially available 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) assay for use in the quantitation of individual tear proteins, pooled human tear proteins, and protein extracted from ex vivo lotrafilcon A, balafilcon A, and etafilcon A contact lens materials. Methods: Ex vivo contact lens extracts, pooled human tears, and individual tear proteins (human serum albumin (HSA), bovine lactoferrin, human secretory immunoglobulin A (sIgA), human lysozyme) were subjected to three solid-phase assays: AB on polyvinylidene difluoride (AB on PVDF) and AB on nitrocellulose (AB on NC) and the CBQCA assay. Micro-bicinchonic acid (micro-BCA) assay was also employed with lens extracts to determine total protein concentration. Individual and pooled tear proteins were referenced to a micro version of the quantitative ninhydrin protein assay. Results: The CBQCA demonstrated the greatest overall sens...

Kinetic measurements of protein conformation in a microchip.

Abstract: This paper presents a microchip-based system for collecting kinetic time-based information on protein refolding and unfolding. Dynamic protein conformational change pathways were studied in microchannel flow using a microfluidic device. We present a protein-conserving approach for quantifying refolding by dynamically varying the concentration of the chemical denaturants, guanidine hydrochloride and urea. Short diffusion distances in the microchannel result in rapid equilibrium between protein and titrating solutions. Dilutions on the chip were tightly regulated using pressure controls rather than syringe-based flow, as verified with extensive on-chip tracer dye controls. To validate this protein assay method, folding transition experiments were performed using two well-characterized proteins, human serum albumin (HSA) and bovine carbonic anhydrase (BCA). Transition events were monitored through fluorescence intensity shifts of the protein dye 8-anilino-1-naphthalenesulfonic acid (ANS) during dilutions of protein from urea or guanidine hydrochloride solutions. The enzymatic activity of refolded BCA was measured by UV absorption through the conversion of p-nitrophenyl acetate (p-NPA). The microchip protein refolding transitions using ANS were well-correlated with conventional plate-based experiments. The microfluidic platform enables refolding studies to identify rapidly the optimal folding strategy for a protein using small quantities of material.

[Structural analysis and anti-tumor activity in vivo of polysaccharide APS-2a from Angelica sinensis].

Abstract: The polysaccharide APS-2a was isolated from Angelica sinensis (Oliv.) Diels through water extraction, deprotein, ethanol precipitation and DEAE-sephades A-25 column chromatography respectively,and was further purified by Sephacryl S-400 and Sephadex G-100 column chromatography. The phenol-sulfuric acid assay and Bradford method were used to determine the contents of carbohydrate and protein, respectively. The molecular weight was carried out with high-performance size exclusion chromatography (HPSEC) combined with a differential refractometer detector. The monosaccharide compositions were determined by gas chromatography after complete hydrolysis with acid. The models of mice transplanted sarcoma S-180 were used to study the anti-tumor effects in vivo. Thymus indexes, spleen indexes were determined. The HPSEC result showed the APS-2a was a single homogeneous component and its weight average molecular weight was 7.4 x 10(5) Da. The monosaccharide composition of APS-2a was glucose, galactose, arabinose, rhamnose, galcturonic acid. Furthermore, APS-2a (20.50 mg/kg) could inhibit the proliferation of tumor cells in mice transplanted S-180. The thymus indexes and spleen indexes in the groups treated with APS-2a were higher than control group.

Adsorption of peanut (Arachis hypogaea, Leguminosae) proteins by activated charcoal.

Abstract: The binding of peanut protein allergens to activated charcoal (AC), used medically for gastric decontamination following the ingestion of toxic substances, was investigated for potential clinical application. Crude peanut extract (CPE) or purified peanut protein allergens Ara h 1 and 2 were co-incubated with AC under a variety of conditions followed by centrifugation to remove the AC and adsorbed protein. The resulting supernatant solution was analyzed for unadsorbed protein by gel electrophoresis and quantitative protein assay. The extent of protein adsorption by a known amount of AC was determined. Protein binding to AC was rapid and irreversible. The extent of adsorption was unaffected by pH, but was optimal near physiological salt concentrations. Denatured proteins, or those of larger molecular weight, required more AC than smaller or native proteins. The extent of protein binding increased with temperature, supporting the concept that protein molecules diffuse into vacant pores of appropriate size on the charcoal surface.

A simple stained protein assay (SPA) for rapid total protein determination applied to spinal fluids.

Abstract: A rapid, simple technique measures small amounts of total protein applied on an agarose gel slab. Alternating current through the gel may reduce diffusion of the protein, which is chemically fixed. The intensity of the Coomassie-Blue-stained spots is a function of the amount of protein determined by comparison with simultaneously run standards. Densitometric and visual estimates of protein in spinal fluids by the stained protein assay correlated with Lowry values highly significantly (n = 28; P less than 0.001, Spearman test of rank correlation). As little as 0.2 mug protein was measured; this figure should be compared with 10 mug for the Lowry method. The reproducibility of the method was +/- 5%.

Method for detecting total foam protein content of beer barley and malt

Abstract: The present invention provides a method for testing the general foam protein in the beer barley and the malt The present invention adopts an optimized albumen extracting method, uses the popular and common Coomassie brilliant blue ration rationed albumen method (Bradford method) to test the change of the general foam protein in the beer barley and malt The method is simple and quick, and has strong applicability

Effects of different cell lysis buffers on protein quantification

Abstract: OBJECTIVE To observe the effects of different cell lysis buffers on protein quantification with Bradford method and bicinchoninic acid (BCA) method. METHODS Bradford method and BCA method were used to determine the concentration of bovine serum albumin (BSA) in different solutions (distilled water, cell lysis buffer used in two-dimensional differential in-gel electrophoresis and three kinds of cell lysis buffers used in conventional two dimensional gel electrophoresis), as well as the protein concentrations of cell lysates using these different lysis buffers. Bradford method was also applied to determine the protein concentrations of samples with repeated freeze thaw cycle, in different colorimetric cylinders, or using different standard curves from different periods. RESULT The protein measurements increased for 1.2 to 2 fold when different cell lysis buffers were used in Bradford method, but the measurements increased with the increased concentration of BSA (r=0.989 approximately 0.996, P<0.05). For BCA, measurement reading increased about thousands times higher, even overflowed the limits of machine. Protein measurements didn't change significantly, only showed a declined trend after repeated freeze thaw cycle, while no significant changes were found using different colorimetric cylinders or standard curves from different periods. CONCLUSION Bradford method may be the choice of the protein quantification in proteomics. However, optimization is required for specific experimental conditions.

Determination of Bovine Serum Albumin by Resonance Light Scattering Technique

Abstract: In this paper,the bovine serum albumin(BSA)-β-mercaptoethanol(β-ME)-sodium dodecylbenzene sulphonate(SDBS) system was studied by resonance light scattering technique.Based on the RLS intensity difference between β-ME-SDBS system and BSA-β-ME-SDBS system,a simple assay for BSA was developed.The influences of some experimental factors,including pH value,concentration of β-ME and SDBS,addition sequence of reagents and foreign substances,on the enhancement of the RLS intensity were studied.Under the optimal conditions,the enhanced RLS intensity was proportional to the concentration of BSA in the range from 1.0×10-8 mol\5L-1 to 7.0×10-7 mol\5L-1 with the detection limit of 6.0×10-9 mol\5L-1.Some metal ions had little effect on the determination of BSA.The results of assay for BSA in synthetic samples and real sample were satisfactory,which were in good agreement with the results obtained by Bradford method.

Physicochemical Properties and Anti-tumor Activity in vitro of Polysaccharide APS-3c from Angelica sinensis(Oliv.) Diels

Abstract: The polysaccharide APS-3c was isolated from Angelica sinensis(Oliv.) Diels through water extraction,deprotein,ethanol precipitation,and DEAE-column chromatography respectively,and was further purified by Sephacryl S-400 and Sephadex G-100 column chromatography.The phenol-sulfuric acid assay,carbazole method and Bradford method were used to determine the contents of carbohydrate,galacturonic acid and protein,respectively.The molecular weight was carried out with high-performance size exclusion chromatography (HPSEC) combined with a differential refractometer detector.The monosaccharide compositions were determined by gas chromatography after complete hydrolysis with acid.The HPSEC result showed that the APS-3c was a single homogeneous component and its molecular weight was 1.4×104 Da.The monosaccharide composition of APS-3c was arabinose,rhamnose,glucose,galactose,mannose,xylose,galacturonic acid.The results of the MTT assay indicated that APS-3c inhibited the proliferation of human myeloblastic leukemia HL-60 cells in a concentration-dependent manner.The inhibition rate of 10 mg/L APS-3c on human colon carcinoma SW1116 cells was 35.1%.

Biochemical characterization of a xylanase from Paenibacillus sp. HPL-001 expressed in Escherichia coli

Abstract: Xylanase (EC 3.2.1.8) is the name given to a class of enzymes which degrades the linear polysaccharide beta-1,4-xylan into xylose, thus breaking down hemicellulose, which is a major component of the plant cell wall. A new xylanase gene, KRICT PX1 isolated from Paenibacillus sp. HPL-001, was expressed in E. coli, and the biochemical properties of the enzyme was examined. The GSTfused xylanase was purified with immobilized glutathione column, and xylanase activity of the purified GST-fused protein was 10.97 U/mg protein with Km value of 3.16 for xylan from birch wood. The optimum pH and temperature for the activity of the enzyme were 3~4 and 40~50°C, respectively. Most salts, such as NaCl, LiCl, KCl, NH4Cl, CaCl2, MgCl2, MnCl2, and CsCl2 did not change the enzyme activity at 1 mM except CuSO4, ZnSO4, and FeCl3. And also, 1 mM of ethylenediamine tetra-acetic acid (EDTA), 2-mercaptoethanol (ME), and phenylmethanesulphonyl fluoride (PMSF) were not effective on the enzyme activity. The enzymatic product resulting from the reaction with birch wood xylan was mostly xylose with a small amount of arabinose. MATERIALS and METHODS Paenibacillus sp. HPL-001 (KCTC11365BP) SEM Photograph TSA Medium Xylan Overlaid The Xylanase, KRICT PX1 with 332 AAs ► gDNA Library construction (1,536 clones) with 5kb DNAs from HPL 001 whole genome pCB31 Plasmid vector insertion & transformed to E.coli DH10B ► Xylan degrading clone screening 96-well DNS assay for xylose & xylan-overlaid agar-plate assay ► New xylanase (KRICT PX1) identification Based on the results from sequencing and ORF analysis, KRICT PX1 has the homology of 70% with AAP51133-1, 68% with EDS52673-1, and 67% with ABI49937-2 from ‘Blast P’ search GST-Fused KRICT PX1 Protein T7P = T7 Promoter, RBS = Ribosomal binding site, GST = glutathione-S-transferase protein, Xa = Factor Xa restriction protease cleavage site, MCS = Multiple cloning site for the insertion of the target gene, T7T = T7 Terminator ► GST-Fused KRICT PX1 separation E. coli BL21-Gold (DE) with pIVEX-GST-KRICT PX1 collapsed by sonic cell disruption The protein separated by GST-binding resin (Novagen) and eluted with glutathion ► Protein determination, xylanase assay, & electrophoresis Bradford assay for total protein determination with a BSA as standard protein DNS (3,5-dinitrosalicylic acid) assay for the released xylose from birch wood xylan at 540 nm GST-fused KRICT PX1 protein (MW 65kd) confirmed with SDS-PAGE by Laemmli method ► Biochemical property of GST-Fused KRICT PX1 Xylanase activity at the range of pH 4~10, and temperature at 20~80°C examined The effect of metal ions and additive chemicals on the activity examined Michaelis constant, Km was evaluated with the substrate of birch wood xylan Enzymatic products of xylan was analyzed with HPLC (mobile phase ACN :water=80 : 20, Shodex NH2P-50 4E column, ELSD detector) CONCLUSIONS