Showing papers on "Bradford protein assay published in 2018"

Quantifying Bound and Active Antibodies Conjugated to Gold Nanoparticles: A Comprehensive and Robust Approach To Evaluate Immobilization Chemistry

Abstract: Gold nanoparticles (AuNPs) functionalized with antibodies have the potential to improve biosensing technology because of the unique optical properties of AuNPs and the specificity of antibody-antigen interactions. Critical to the development and optimization of these AuNP-enabled sensing technologies is the immobilization of the antibody onto the AuNP. The development of novel immobilization strategies that optimize antibody loading and orientation in an effort to enhance antibody activity, and therefore assay performance, has been the focus of many recent studies. However, few analytical methods exist to accurately quantify the activity of conjugated antibodies and reliably compare different immobilization strategies. Herein, we describe an enzyme-mediated assay to quantify the fraction of the immobilized antibodies that is accessible for antigen binding. Anti-horseradish peroxidase (anti-HRP) antibody is mixed with AuNPs to allow for conjugation, and the unbound, excess antibody is quantified with a modified Bradford assay to determine antibody loading onto AuNPs. The conjugates are then mixed with excess HRP to saturate all accessible binding sites, and bound HRP is quantified based on enzymatic reaction rate. This analytical scheme was used to compare two common immobilization strategies, nonspecific adsorption and protein A-mediated immobilization. We found that the antibody surface coverage is greater for direct adsorption than protein A-mediated binding; however, 23 ± 6% of the directly adsorbed antibodies were active, whereas 91 ± 19% of the antibodies bound through protein A were active. In addition to establishing this method as quantitatively precise and accurate, our results emphasize the need to quantify both antibody loading and antibody activity upon conjugation to gain greater insight into differences in immobilization chemistries and identify optimum protein conjugation strategies to maximize immunoassay performance.

Optimization of the cydex blue assay: A one-step colorimetric protein assay using cyclodextrins and compatible with detergents and reducers.

Abstract: Sodium dodecyl sulfate electrophoresis (SDS) is a protein separation technique widely used, for example, prior to immunoblotting. Samples are usually prepared in a buffer containing both high concentrations of reducers and high concentrations of SDS. This conjunction renders the samples incompatible with common protein assays. By chelating the SDS, cyclodextrins make the use of simple, dye-based colorimetric assays possible. In this paper, we describe the optimization of the assay, focussing on the cyclodextrin/SDS ratio and the use of commercial assay reagents. The adaptation of the assay to a microplate format and using other detergent-containing conventional extraction buffers is also described.

Affinity Binding-Induced Hg2+ Release and Quantum Dot Doping for General, Label-Free, and Homogenous Fluorescence Protein Assay.

Abstract: Herein, a general protein conversion and analysis strategy was developed for homogeneous, label-free, and sensitive protein detection, on the basis of the affinity binding-induced Hg2+ release for protein conversion, and the succeeding Hg2+ doping-induced ZnSe quantum dot (QD) photoluminescence for signal readout. Two DNA motifs were designed, each of which was conjugated with a protein-specific recognition ligand. The mercury ions were initially introduced into one DNA motif by T-Hg2+-T interaction. The Hg2+ releasing was then accomplished after protein recognition-initiated strand exchange reaction between two DNA motifs. Then, the simultaneous incorporation of the released Hg2+ into ZnSe QD resulted in a doping-dependent fluorescence emission at 560 nm correlated with protein analysis. The protein assay is outperformed only by a simple one-step mixing operation but no separation or washing steps. Also, the use of doped QD as a fluorogenic reporter can avoid the fluorophore and/or quencher labeling, and...

An extraction free modified o-phthalaldehyde assay for quantifying residual protein and microbial biofilms on surfaces.

Abstract: Biological contamination of surfaces in industry and healthcare is an important vector of disease transmission. Current assays for detecting surface-adherent contamination require extraction of biological soil. However, physical inaccessibility or poor solubility may limit recovery. Here, how the o-phthalaldehyde (OPA) protein assay can be modified to measure residual protein (modeled with bovine serum albumin) or biofilm on a surface without extraction is described. The assay limit of detection (LOD) for protein was 1.6 µg cm-2. The detection threshold for Staphylococcus epidermis biofilm was 117 µg cm-2. The clinical utility of the method was demonstrated for measurements taken from clinically used endoscopes. Since this method is more sensitive than extraction-based testing, clinical results should not be compared with conventional benchmarks. By enabling direct detection and quantification of soils in complex or hard-to-reach areas, this method has potential to improve the margin of safety in medical and industrial cleaning processes.

Food protein: food colour interactions and its application in rapid protein assay.

Abstract: SaeedS�MG,�AbdullahSU,�SayeedSA,�AliR�(2010):�Food protein: Food colour interactions and its application in rapid protein assay CzechJ�FoodSci,�28:�506-513 Theuniformdistributionofcoloursasadditivesinamajorityofthefoodsystemsisareliableindicationthatoneor� morecomponentsoffoodsareabletobindwithcolourmoleculesandactastheircarriers �However, �thefoodcom- ponentsactingasthecolourcarriershavenotbeenidentified �Thepresentpaperdescribesthebindingcapacityof� Carmoisinewithavarietyoffoodproteins, �ourresultshaveshownthattheintensity, �staining, �andsharpnessofthe� stainedproteinbandswereexcellentascomparedtoCoomassieBrilliantBlueR�250,�whichisanestablishedstain - ingagentforvisualisingelectrophoreticallyresolvedproteins �ThedataillustratesthatCarmoisineisafastreacting� dyeformingcolour-complexeswithalltypesoffoodproteinsincludingcurryleavesproteins �Theproteinbandsare� visualisedwithinanhourwhichisusefulfortheinitialimmediateproteinidentifications�Theexperimentsrelatedto� thestainingoftheresolvedproteinswithCarmoisinehaveshownthatthedyeishighlysensitive, �rapid, �andlasting� Thefood-dyecanprovideaquickproteinassayasoftendesiredinresearchworks, �theresultsmaybelaterconfirmed� byusingCoomassieifsorequired �Inviewofitsstrongbindingwithalmostallproteins, �itwasthoughtthathuman� proteasespresentinthedigestivetractmaynothydrolysetheboundproteinscompletelyandmayrestricttheproteo - lyticdigestion �However, �theexperimentsbasedonthetrypticdigestibilityin vitro revealedthatcolourbindinghas� noadverseeffectonhydrolysisofpeptidebondsbytheintestinalproteases

Reliability of Analytical Methods for Recombinant Human Insulin Quantification in the Bulk Crystals and in-Process Control -

Abstract: The aim of this study was to demonstrate the reliability of the protein determination methods used in the process of recombinant human insulin development before its scale up. The total protein content was measured by Bradford, molar extinction coefficient, and dry weight methods. The standards were analyzed using Mono-Q, Aquapore RP300, and Kromasil columns to calculate the concentrations of the proteins using the theoretical extinction coefficient and peak area. The following highly purified standards were used: batches B4-258 and QS009-010 of sulfonated fusion protein; batches B4-267, B4-268, RALF-018, HGUT-042, HGUT-043, and HGUT-045 of renatured fusion protein; the United States Pharmacopeia reference; and batch B4-253 of bulk insulin crystals. The results were analyzed using ANOVA or Student’s t-test at 95 % significance. The Bradford method showed up to 60 % variation for all evaluated standards, while the remaining two methods were consistent with each other. The chromatographic parameters were used to validate the analytical methods, and all results met the current guidelines of Brazilian regulatory agencies. The use of quality parameters and the statistical evaluation of the data demonstrated that analytical methods used in the in-process control are suitable for the intended purpose, which certifies the reliability of the generated data.

Specific glycine to alanine mutation eliminates dynamic interaction of polymeric GlyT1a N-terminus with Coomassie Brilliant Blue G-250.

Abstract: We previously found that multimeric GlyT1aN16 protein exhibits increased diffusion in a polyacrylamide gel and shows an unusual time-dependent absorbance rearrangement, as revealed by the Bradford assay. Here, we find that glycine to alanine mutation eliminates the absorbance shift, but not the altered diffusion properties of GlyT1aN16, indicating that these two phenomena are not interconnected. The absorbance shift is apparent with both native and urea-denatured GlyT1aN16, suggesting that the effect is either not dependent on protein structure, or the required structure is restored very quickly following denaturant removal. In the far-UV spectra, circular dichroism (CD) curves for both wild-type and mutated GlyT1aN16 are under the zero line, indicating largely unstructured character. However, a significant shift of the mutant CD curve suggests possible microstructural heterogeneity. Deconvolution of the CD data indicates a potential 3-fold increase in isolated helical content, which would inhibit an absorbance shift, as we demonstrated previously. These results suggest that, in addition to protein quantification, Coomassie Brilliant Blue G-250 can be used to reveal certain properties of the secondary structure of proteins.

Applicability of protein estimation methods for assaying glycomacropeptide

Abstract: Glycomacropeptide (GMP) is a nutraceutical additive, which is released during cheese preparation. In this study, the protein content in a laboratory GMP preparation and two commercial GMP samples, which was estimated by the Bradford, biuret, Lowry and bicinchoninic acid (BCA) assays, were compared using the Kjeldahl method. There were apparent differences in the values of estimated proteins using the different methods. An absence of dose response in the Bradford method and a low dose response using the Lowry method indicated the unsuitability of both these methods. Absorbance in the biuret and BCA methods linearly increased with increasing GMP content. The BCA method was the most sensitive of these methods.

Establishment of the collection, storage and preservation methods and their influence on stability of human salivary exosome

Abstract: The functions displayed by exosomes derived from saliva and other body fluids have been established This paper studied the stability of human salivary exosome beginning from the collection mode, storage, and its preservation methods Unstimulated saliva samples were collected from healthy subjects Protease inhibitor was added into each samples and stored under different temperatures and at varying periods of time The exosomes were isolated by ultracentrifugation and confirmed by using Western Blot Exosome morphology was inspected by Scanning Electron Microscope (SEM) and the protein concentration was determined using the Protein (Bradford) Assay The exosome particle size distribution and concentration were calculated using Nanoparticle Tracking Analysis (NTA) The protein assay showed no significant differences in the exosome protein concentration values for all conditions Western Blot analysis also showed no differences in the presence of exosome and all the samples were positive for protein CD63 SEM analysis showed the fine shape of exosome which is round, in vesicle form with the size ranging between 10 nm and 100 nm NTA determined the individual mean and the clumping exosome size was 203 nm Human salivary exosomes remained intact in the absence of protease inhibitor and in different storage temperatures

Detection of Oxidative Stress in Buccal Cells using iSWAB Tubes

Abstract: Oxidative stress is the result of an imbalance in pro-oxidant/antioxidant homeostasis in favor of oxidants leading to the oxidation of DNA, lipids, and proteins. In this experiment, we investigated the oxidative profile of iSWAB-Mawi protein tubes by examining their ability to detect the protein oxidation biomarkers: nitrotyrosine and S-nitrosocysteine in buccal cells in a sample of 40 participants, equally divided between Lebanese University and Benta Pharma Industries. Protein concentrations were determined by Bradford assay in order to then be analyzed for the presence of nitrotyrosine and S-nitrosocysteine by western blot. Results showed the presence of S-nitrosocysteine and nitrotyrosine in buccal cells reflecting the ability of iSWAB-Mawi protein tubes to detect those protein oxidation biomarkers.

Characterization of cellulase from E. coli BPPTCC-EGRK2

Abstract: Cellulase enzymes are widely used in various industries such as detergent industry, bioethanol, animal feed, textile and paper This research focused on characterization of cellulase produced from Eschericia coli BPPT-CC EgRK2, which is a recombinant that can produce protein enzymes endo- β-1,4-glucanase Eschericia coli BPPT-CC EgRK2 was cultured in 1 litre liquid medium Luria Bertani Because the bacteria is intracellular, sonication is needed for cell disruption to get the cellulase enzyme The enzyme activity was then analyzed by CMC substrate at different concentration The protein content analysis was carried out using Bradford method; the molecular weight analysis was done using SDS-PAGE; while the enzyme kinetics was plotted using Michaelis-Menten model Our results showed the highest enzyme activity was 2403 U/ml and the protein concentration was 5352 mg/ml The Michaelis-Menten constant (Km) and maximum velocity (Vmax) for CMC substrate hydrolysis were 007 μmol/ml and 249 μmol/ml/sec, respectively The cellulase molecular weight was 58 kDa using SDS-PAGE with 75% stacking gel The results indicated that Eschericia coli BPPT-CC EgRK2 is a promising renewable source for cellulase production for industrial application

Biuret and Bradford Methods Suitability for Protein Quantification in Rapeseed Meal

Abstract: Being attractive for insects, non-genetically modified rapeseed is valuable for maintaining environmental biodiversity. Primarily, the rapeseed is an important industrial crop which is used for production of vegetable oil. Oil extraction from rapeseeds results in the generation of substantial amounts of rapeseed meal which is used either as a protein rich feed additive or as a source for preparation of protein containing ingredients for food industry. Both applications require frequent evaluation of protein content. Although Kjeldahl method is considered standard, it is not appropriate for routine evaluation of protein content in protein extracts. The aim of the study was to evaluate suitability of biuret and Bradford methods for protein quantification in rapeseed meal extracts. After consecutive triple extraction of proteins with water, 5% NaCl, 70% ethyl alcohol and 0.1 N NaOH, protein evaluation of each albumin, globulin, prolamin and glutelin extraction aliquot demonstrated overall lower protein content by Bradford method compared to biuret method. The most pronounced differences in protein content were observed with prolamin fraction where three fold higher protein concentrations in each extraction aliquot was observed when biuret method was applied for the evaluation. Comparative quantification of the total protein of each of the four fractions followed a similar trend of lower protein content evaluation by Bradford method. Overall results indicated biuret method as more suitable for protein quantification in rapeseed meal extracts which was confirmed by comparison with data obtained by Kjeldahl method.

Quantity and Functionality of Protein Fractions Isolated from 3 Ecotypes of Indigenous Chicken in Kenya

Abstract: The aim of this study was to evaluate the effect of the cluster ecotype and the part of chicken on nutritional composition, and functionality of sarcoplasmic and myofibrillar proteins that are most relevant to the technological features of chicken meat. Over 50 chickens from each ecotype cluster purchased, slaughtered and the meat stored under refrigeration at -20 o C and later on transferred in cooler box on ice and flown to South Africa, at the Durban University of Technology. Protein fractions were extracted with a cocktail of Sodium Chloride buffer (50mM NaCl, 50mM Tris HCl; 75mM DTT and 1mM EDTA at pH 7) and quantified by Bradford method. One dimensional Sodium Dodecyl Polyacrylamide Gel Electrophoresis (SDS PAGE) was applied to separate protein fractions. Emulsifying capacity, emulsifying stability, solubility, and in vitro digestibility were determined on the total protein isolates. Significant differences in band expressions were recorded for the myofibrillar and the sarcoplasmic proteins. The three ecotypes had high quality proteins with all the limiting and essential amino acids at concentrations higher than FAO/WHO recommended daily allowance for adults and children. Distinct protein bands at larger molecular weight proteins >100 kDa, corresponding to Myosin Heavy Chain, medium fractions 75 kDa and 45 kDa and even lower molecular weight fraction <25 kDa were present in the chicken breast and the thighs. It concludes that Indigenous chicken protein isolates’ nutritional and functional properties are affected by part of chicken and ecotype clusters.

Denaturation of Lysozyme with Visible-light-responsive Photocatalysts of Ground Rhodium-doped and Ground Rhodium-antimony-co-doped Strontium Titanate.

Abstract: Protein denaturants play an important role in medical and biological research, and development of new denaturants is widely explored to study aging and various diseases. In this research, we treated lysozyme, a model protein, with photocatalysts of ground Rh-doped SrTiO3 (g-STO:Rh) and ground Rh-Sb-co-doped SrTiO3 (g-STO:Rh/Sb) under visible light irradiation to explore the potential of those photocatalysts as denaturants. SDS-PAGE showed that photocatalysis with g-STO:Rh induced the fragmentation of lysozyme into unidentifiable decomposition products. BCA and Bradford protein assays indicated that the peptide bonds and basic, aromatic and N-terminal amino acid residues in lysozyme were denaturated by g-STO:Rh photocatalysis. The denaturation of those amino acids, as quantified by the decreased solubility of lysozyme, was estimated to be more severe by Bradford protein assay than by BCA protein assay. Circular dichroism (CD) spectra of lysozyme revealed that the secondary structure was denatured by g-STO:Rh photocatalysis, indicating that g-STO:Rh photocatalysis is especially effective against the amino acid residues that form the secondary structure via hydrogen bonds. Furthermore, the lytic activity of lysozyme was reduced by g-STO:Rh photocatalysis, owing to denaturation of the enzyme. The visible-light-responsive photocatalyst of g-STO:Rh/Sb accelerates the oxidation reaction and has stronger oxidizing power than g-STO:Rh. Lysozyme was denatured more quickly by g-STO:Rh/Sb photocatalysis than by g-STO:Rh according to analysis by SDS-PAGE, CD spectroscopy, BCA and Bradford protein assays, and lytic activity. These results suggest that higher photocatalytic activity induces more significant denaturation of lysozyme, implying that the main factor of photocatalytic denaturation of lysozyme is oxidation. It should be noted that, as far as we know, this is the first report for denaturation of protein using visible-light-responsive photocatalyst.

The Antimicrobial Properties of Honey and Their Effect on Pathogenic Bacteria

Abstract: The Antimicrobial Properties of Honey and Their Effects on Pathogenic Bacteria Shreena Himanshu Mody Department of Microbiology and Molecular Biology, BYU Master of Science Honey has been used to heal wounds since ancient times and there are many references in ancient literature that cite honey for its medicinal uses. It is used as an alternative agent to cure infections of wounds, burns, ulcers etc. Researchers have shown some of the antimicrobial properties of honey when used as an ointment. The purpose of this study was to examine the antimicrobial properties of honey from Utah and other locales, and to identify promising antimicrobial activities that could be useful in treating infections caused by resistant bacteria. Five different bacteria and eight different honey samples were used. To see the effects of honey on bacteria, various methods were employed. A disk diffusion assay was used to measure zones of inhibition. Osmolarity was measured to examine total solute differences. An Amplex® Red hydrogen peroxide/peroxidase assay kit was used to measure the amounts of hydrogen peroxide in the various honey samples. Protein assays were performed to examine total protein content and also to identify the presence of known antimicrobial proteins. The pH of each honey sample was also measured. Honeys used in this study showed relatively similar sugar contents and pH levels. One honey sample, NY, did not show any antimicrobial activity when it was tested against several bacterial pathogens. It also possessed a lower content of protein and hydrogen peroxide. Major Royal Jelly Protein 1 (MRJP1) was found in abundance in all honey samples. Sample 13 showed good antimicrobial activity even though it had lower concentrations of hydrogen peroxide than sample 14 and M+20. Sample 13 also had a slightly lower protein content than the other samples that displayed significant antimicrobial activity. The catalase inhibition studies showed that sample 13 displayed significant hydrogen peroxide activity. A more detailed study of the antimicrobial properties of these components may lead to the identification of useful therapeutics that can be used in our never-ending war against microbial infections.

Novel method for enriching protein sample from used cooking oil sample and detecting content of protein sample

Abstract: The invention provides a method for enriching a protein sample from a used cooking oil sample and detecting the content of the protein sample. A pretreatment method for enriching protein from grease of used cooking oil is established by screening and verifying different methods for enriching protein from grease; the solubility of fat-soluble protein is improved by utilizing tetrahydrofuran as a solvent based on the Bradford method for detecting the protein content, and a detection method for detecting the protein content from a used cooking oil sample is established.

Purification and characterisation of protein from natural rubber latex (NRL) skim serum

Abstract: NRL wastewater contains Hevea proteins. Proteins purified from skim serum of latex concentrate processing plant underwent physical characterisation such as yield and pH, spectroscopy (FTIR and UV) and SDS PAGE. This work aims to purify this hev proteins and further purify to confine specific hevs based on molecular weight eg. hevein. The waste were conditioned with various pH before precipitation. Highest yield were attained at lower pH condition. The chromatographic profile obtained via UV spectrophotometer at 280 nm absorbance and the protein concentration was estimated via UV at 590 nm using Bradford assay kit. Photometric dispersion analyzer and UV at 280 nm were used to construct chromatographic profile. The objectives of fractionating those hev proteins were to get specific proteins of interest such as hevein hev b 6 and Manganese superoxide dismutase hev b10 but SDS PAGE analysis showed a few bands in one fractions. The results indicated incomplete fractionation and the amount collected was too small. Thus, the study on systematic reduction in load amount was conducted to scale up open column for future plan.