Showing papers on "Bradford protein assay published in 2007"

Bifunctional Au-Fe3O4 nanoparticles for protein separation.

Abstract: In this article, we report the synthesis of bifunctional Au-Fe(3)O(4) nanoparticles that are formed by chemical bond linkage. Due to the introduction of Au nanoparticles, the resulting bifunctional Au-Fe(3)O(4) nanoparticles can be easily modified with other functional molecules to realize various nanobiotechnological separations and detections. Here, as an example, we demonstrate that as-prepared Au-Fe(3)O(4) nanoparticles can be modified with nitrilotriacetic acid molecules through Au-S interaction and used to separate proteins simply with the assistance of a magnet. Bradford protein assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were performed to examine the validity of the separation procedure, and the phosphate determination method suggested that the as-separated protein maintained catalytic activity. This result shows the efficiency of such a material in protein separation and suggests that its use can be extended to magnetic separation of other biosubstances. Moreover, this synthetic strategy paves the way for facile preparation of diverse bifunctional and even multifunctional nanomaterials.

Assays for Determination of Protein Concentration

Abstract: Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels. © 2016 by John Wiley & Sons, Inc.

Phenol extraction of proteins for proteomic studies of recalcitrant plant tissues.

Abstract: Phenol extraction of proteins is an alternative method to classical TCA-acetone extraction. It allows efficient protein recovery and removes nonprotein components in the case of plant tissues rich in polysaccharides, lipids, and phenolic compounds. We present here a tried and tested protocol adapted for two dimensional electrophoresis (2-DE) and further proteomic studies. After phenol extraction, proteins are precipitated with ammonium acetate in methanol. The pelleted proteins are then resuspended in isoelectric focusing buffer, and the protein concentration is measured with a modified Bradford assay prior to electrophoresis. The important points for successful use of this protocol are (1) keeping samples at very low temperature during the first step and (2) careful recovery of the phenolic phase after the centrifugations, which are major features of this protocol.

Assays for determination of protein concentration.

Abstract: Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This unit describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the unit is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This unit also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

Magnetic-Core/Porous-Shell CoFe2O4/SiO2 Composite Nanoparticles as Immobilized Affinity Supports for Clinical Immunoassays†

Abstract: This study demonstrates a novel approach towards the development of advanced protein assay systems based on physically functionalized, magnetic-core/porous-shell CoFe 2 O 4 /SiO 2 composite nanoparticles. The preparation, characterization, and measurement of the relevant properties of the protein assay system is discussed, and the system is used for the detection of cancer antigen 15-3 (CA 15-3, used as a model here) in clinical immunoassays. The protein assay system, based on nanometersized magnetic cores and silica shells, shows good adsorption properties for the selective attachment of CA 15-3 antibodies specific to CA 15-3. The core/shell nanostructures exhibit good magnetic properties, which enables their integration into a quartz crystal microbalance (QCM) detection cell with the help of a permanent magnet. Under optimal conditions, the resulting immunoassay system presents a good QCM response for the detection of CA 15-3, and allows the detection of CA 15-3 at concentrations as low as 1.5 U mL -1 (U: units). Importantly, the proposed protein assay system can be extended to the detection of other antigens and biological compounds.

Polyphenolic compounds interfere with quantification of protein in soil extracts using the Bradford method

Abstract: The Bradford protein quantification assay is based on an absorbance shift in Coomassie brilliant blue G-250 (CBB). Samples extracted for glomalin, a protein produced by arbuscular mycorrhizal (AM) fungi, are quantified using the Bradford assay. CBB is known to react with polyphenolic substances, and co-extraction of glomalin and humic substances is known to occur. The effects of increasing concentrations polyphenolic compounds were measured. The addition of any amount of polyphenolic compounds increased the Bradford reactive fraction (BRF) of soil extract. Caution is required when interpreting BRF data, as comparison of BRF data from different studies or different field sites is problematic. The BRF may represent recalcitrant organic material in soil, though its relationship to AM fungi remains unclear.

Application of a modified Coomassie brilliant blue protein assay in the study of protein adsorption on carbon thin films

Abstract: The aim of this study is to investigate the adsorptive property of proteins on surfaces of three kinds of carbon films (DLC, CN 0.088 , and CN 0.15 ) and the medical origin of PMMA. The carbon films were first synthesized by magnetron sputtering with different deposition parameters. The surface characteristics of both the carbon films and the original samples were then measured using a contact-angle meter. A modified Coomassie brilliant blue (CBB) protein assay (or Bradford method) was used to detect the amount of proteins adsorbed on the material surfaces after the pilot research for the optimal ratio of protein- to CBB-solution had been performed. The results of the pilot research show that the optimal ratio of protein- to CBB-solution is 1:3, and the protein adsorption results show that the sequence amounts of proteins adsorbed on four kinds of surfaces are DLC > CN 0.088 > PMMA > CN 0.15 . This was the same as the hydrophobicity sequence of four kinds of surfaces obtained from the contact-angle test. However, the ratio of albumin to IgG ( R A/I ) adsorbed on the four surfaces has an order of PMMA > CN 0.15 > DLC > CN 0.088 , which indicates the anti-thrombogenicity property of the four kinds of surfaces.

Measurements of label free protein concentration and conformational changes using a microfluidic UV-LED method.

Abstract: This paper presents a microchip-based system for measuring concentrations and dynamic conformational changes in proteins without any use of extrinsic fluorescent labeling. The microchannel flow of protein molecules was integrated with an ultraviolet light-emitting diode (UV-LED, lambda ex = 295 nm) and a photodetector (lambda em = 330 nm). The intrinsic fluorescence shift, arising from selectively exciting aromatic amino acid tryptophan (Trp), was monitored to quantify refolding pathways by dynamically varying the concentration of the chemical denaturant, 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. The concentrations of proteins were first measured using the UV-LED microfluidic platform, and the data showed detection limits down to 72, 128, and 250 nM for tryptophan, bovine serum albumin (BSA), and bovine carbonic anhydrase (BCA), respectively. To validate the protein assay method, folding transition experiments were performed using a well-characterized protein, BSA. The microchip protein refolding transitions using intrinsic fluorescence were well-correlated with conventional fluorometer experiments. The microfluidic platform facilitates refolding studies to identify rapidly the optimal folding strategy for a protein using small quantities of material. The technique offers a real alternative to bulky microfluidic systems consisting of large and expensive laser-based designs.

Microwave-enhanced ink staining for fast and sensitive protein quantification in proteomic studies.

Abstract: A novel microwave-enhanced ink staining method was developed for rapid and sensitive estimation of protein content in sample buffers containing chaotropes, dyes, detergents, and reducing agents. Dye-based Blue-Black ink was used to quantitatively visualize proteins spotted on a nitrocellulose membrane. The total staining time was greatly reduced to 3 min by brief exposure to microwave radiation. The stained membrane was washed with distilled water, baked in a microwave oven for complete desiccation, transparentized with mineral oil, and documented by a desktop scanner or densitometer. Only 1 microL of protein sample (protein solubilized in SDS-PAGE sample buffer or IEF rehydration buffer) was used for protein spotting. The novel solid-phase protein assay gives a 500-fold dynamic range from 19.5 to 10000 ng/microL and can be scaled up for high-throughput protein quantification analysis. The fast, sensitive and low-cost microwave-enhanced ink staining procedure is ideal for protein quantification in proteomic analysis.

Effect of ciprofloxacin and chloramphenicol on humoral immune response elicited by bovine albumin encapsulated in niosomes.

Abstract: The aim is to evaluate the effect of ciprofloxacin and chloramphenicol on anti-BSA antibody production triggered by bovine albumin encapsulated in non-ionic surfactant vesicle, niosomes. Reverse phase evaporation method was adopted to entrap the antigen in colloidal carrier composed of Span 80 and Span 85 followed by simultaneous characterization for particle size, entrapment efficiency and in vitro release. The protein content was determined by Bradford method using UV Visible Spectrophotometer at 595 nm. Humoral immune response was measured in terms of systemic IgG antibody titre by ELISA method. Experimental data indicated that 7 : 3 molar ratio of Span 80 and cholesterol based niosomal formulation possessed maximum (39.8 +/- 2.9)% of soluble protein. Ciprofloxacin markedly (P 0.05). It is necessary to explore the effect of a vaccine antigen when a candidate is medicated with a therapeutic agent, which might help in programming a new drug management and vaccination programme.

Continuous aqueous phase extraction of proteins: automated on-line monitoring of fumarase activity and protein concentration

Abstract: Automated assay techniques are described for on-line measurements of fumarase activity and total protein concentration, including in-line sample dilution and sample multiplexing during continuous aqueous phase extraction. Fumarase was determined by following the conversion of L-malate to fumurate at a wavelength of 250 nm, while the protein assay was based on the Biuret reaction. Actual assay times of 2 and 4 min were achieved for the fumarase and protein measurements, respectively, with an effective measurement cycle time of 2 min. Standard deviations of c. 3.2 and 2% of the measured values were calculated for the enzyme and protein values, respectively. The assay system was coupled to a computer to allow on-line data visualization and storage.

Exploiting flow injection system with mini-immunoaffinity chromatographic column for chondroitin sulfate proteoglycans assay.

Abstract: A flow injection (FI) system with a mini-immunoaffinity chromatographic column was used to perform on-line assays of specific proteoglycans. The 300-μL mini-column contained beads coupled with monoclonal antibodies against the specific sulfation pattern of chondroitin sulfate proteoglycans, which have been reported to be a potential biomarker for cancer. The amount of these proteoglycans present was estimated indirectly from their protein content using the Bradford assay, which is an alternative to a direct carbohydrate assay. The system developed was tested by assaying for chondroitin sulfate proteoglycans in sera from patients with various cancers and comparing the results to those obtained for sera from healthy people. The results indicated that this approach could be used as a cost-effective alternative system for determining the amount of these specific biomarker proteoglycans. The column could be reused at least 90 times, with each run consisting of 200 μL of serum sample diluted twofold; an analysis rate of 30 min per run was achieved, as compared to 4 h for a batch procedure.

Isolation and Characterization of Silaffin that Catalyze Biosilica Formation from Marine Diatom Chaetoceros gracilis

Abstract: The method of making silica in industries requires extreme conditions. The finding of proteins involved in the formation of biosilica from diatoms, has opened up an alternative way of production. Chaetoceros gracilis is one of the diatoms, which is potential in producing silaffin protein. This study aimed to isolate and to characterize the protein. We also analyzed the protein activity toward tetraethoxyorthosilicate (TEOS) substrate in in vitro reaction. Diatom biomass was harvested and further kept in 2% SDS/100 mM EDTA solution. Protein isolation was conducted by dissolving the silica and separating the protein by soaking in 2 M HF/8 M NH4F. Protein concentration was analyzed using Bradford method and the molecular weight was estimated through SDS-PAGE. Protein activity was observed by reacting it with TEOS substrate to form silica polymer and measured by colorimetric molibdate assay. Protein concentration was 1.20 mg/ml and appeared filamentous. The apparent molecular weights consisted of 12, 23, 42, 44 kDa. These protein was able to polymerize the silica at room temperature within 10 min. As much as 85.65 umol TEOS was polymerized per 1.4 x 106 silaffin protein per min. SEM analysis showed the formation of spherical, aggregate biosilica. Key words: Chaetoceros gracilis, silaffin protein, biosilica, polymerization

Protein chemistry, peptide mapping, and preliminary structural characterization of saccharomyces cerevisiae sterol c-24 methyltransferase expressed in escherichia coli

Abstract: At this time, no 3-dimensional structure of the Sterol C-24 Methyltransferase (SMT) enzyme has been discovered. Having such a representation of this enzyme, especially with a bound inactivator, will illustrate what contact amino acids and motifs are necessary for catalysis of a methyl transfer from S-adenosyl- L-methionine (AdoMet) to the C-24 position on the preferred substrate of zymosterol in the ergosterol biosynthetic pathway. In order to achieve this goal, protein chemistry techniques of Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE), Q-sepharose anion-exchange, and 26/60 SephacrylTM S-300 gel permeation chromatography were used to generate pure SMT for structure determination by our colleague Dr. David W. Christianson at the University of Pennsylvania. Quantification methods involving Bradford protein assay, Ultraviolet (UV) absorbance at 280 nm, activity assays with [methyl - 3H3] AdoMet, and Western blot analysis were developed to track the amount of total protein and active SMT from the yeast Wild Type (WT) and Y81W mutant throughout the purification process. Peptide mapping, using the mechanism-based inactivator [3-3H]26,27 dehydrozymosterol (DHZ), was also developed to independently locate motifs in the primary sequence associated with the active center. The main experimental findings are as follows: (1) From 5 g of Escherichia coli (E. coli) BL21 (DE3) host cell pellet harboring the pET23a(+) plasmid with the Y81W mutation was obtained 5 mg Fast Protein Liquid Chromatography (FPLC) pure recombinant Y81W mutant SMT-DHZ-AdoMet complex. (2) From a partial tryptic digest of a SMT-DHZ-AdoMet complex was obtained 9.1 mg of DHZ bound peptide. The identity of the peptide-DHZ complex was not resolved. (3) Using a sample of FPLC pure Y81W mutant SMT complexed with DHZ was attempted X-ray diffraction. Poor resolution crystals were obtained and did not diffract well. The results are interpreted to imply the approaches developed herein to purify SMT can be applied to further structural determination.

Study of Protein Adsorption and Conformational Change on DLC and Ti Surfaces Using FTIR and Coomassie Brilliant Blue Protein Assay

Abstract: The aim of present work was to study the interaction between human plasma protein-albumin (Alb) and immunoglobulin G (IgG) and the surfaces of two kinds of diamond-like carbon (DLC-A and DLC-B) and titanium (Ti) film. Fourier transform infrared spectroscopy (FTIR) was used to perform both quantity investigation and secondary structure analysis of above two proteins adsorbed on material surfaces. A modified Coomassie brilliant blue (CBB) protein assay was also used to study the amount of adsorbed proteins. The result of FTIR quantitative evaluation shows that the ratio of adsorbed Alb to IgG (RA/I) on three kinds of material surface has an order: DLC-A > DLC-B > Ti, which is coincide with the result from CBB protein assay. The result of secondary structure analysis shows that the conformation of Alb and IgG changes in a largest degree after adsorbed on Ti and a smallest degree on DLC-A surface. Both the results indicate that the anti-thrombogenicity of DLC-A seems to be the best and Ti is the worst.

[Screening of Bacillus sp. strains producing thermostable alpha-amylase].

Abstract: Fifteen thermostable alpha-amylases have been obtained as a result of screening of 76 Bacillus sp. strains, isolated from different natural substrates. They have shown 30-100% of their activities after 60 min of incubation at 100 degrees C. Seven thermostable a-amylases demonstrated 100% of their activities after 60 min of incubation at 100 degrees C. These enzymes showed maximal activities at pH 9.0 and exhibited 10-30% of activities at pH 11.0 and 12.0. These properties make them promising for future research and possible practical use. In this paper we have proposed a modified iodometric method for measurement and calculation of alpha-amylases activity. The influence of ammonium sulfate on protein concentration measurement by Bradford method was demonstrated and a necessity of dialysis after precipitation with ammonium sulfate was proposed.

A novel method for determination of protein in human serum utilizing the trihydroxylphenylfluorone-molybdenum(VI) complex as a probe by fluorescence spectrometry.

Abstract: The fluorescence intensity of the trihydroxyl-phenylfluorone-molybdenum(VI) [Mo(VI)] complex is quenched by protein. Based on this, a novel method for protein assay in aqueous solution was developed. With pH 3.75 acetic acid-sodium acetate buffer solution, in the presence of p-octyl polyethylene glycol phenyl ether microemulsion, the quenched fluorescence intensity is proportional to the concentration of bovine serum albumin (BSA) in the range of 0-7.00 microg/mL, and the detection limit of BSA is 5.65 ng/mL. There is no interference from amino acids and most metal ions. The method developed in this paper has been used for the successful determination of protein in human serum.

The entrapped efficiency of BSA liposome

Abstract: BSA liposomes were prepared with approximately 100 nm mean particle size under rather gentle experiment conditions, and two-colorimetric coomassie brilliant blue protein was employed to measure the free drug in the entrapped efficiency (EE%) determination of BSA liposomes. Gel filtration was used to measure the EE%, and several Sephadex gels were examined by the separation of liposomes and free drug. To determine the free drug, three methods were compared on two-colorimetric UV spectrophotography, Bradford and two-colorimetric coomassie brilliant blue, separately. Two-colorimetric coomassie brilliant blue process increased the accuracy and improved the sensitivity of the assay about 20-fold comparing with the Bradford method. Two-colorimetric coomassie brilliant blue assay appeared to be more sensitive and showed broader dynamic range to measure the free BSA in the EE% determination of BSA liposome.

Studies on the Toluidine Blue Dimer as a Fluorescence Probe for Protein Assays

Abstract: A spectrofluorometric method was developed for the determination of total serum protein by exploring toluidine blue (TB) as the fluorescence probe The fluorescence intensity of TB at 648 nm was significantly quenched in the presence of sodium dodecylbenzene sulfonate (SDBS) by forming a dimer of the dye, which can afterwards reconvert to monomer when proteins were added accompanied by the recovery of the fluorescence This might be attributed to the modulated transferring of the dimer‐monomer equilibrium of TB in the anionic surfactant caused by the addition of protein A linear calibration graph was obtained in the range of 05–50 mg/l BSA, with a detection limit of 015 mg/l and a RSD of 13% (n=11, 50 mg/l BSA) Total proteins in human serums were analyzed by using the present procedure and the results agreed well with those obtained by the Biuret method

A Possible Role of Peptides in the Growth Enhancement of an Industrial Strain of Saccharomyces sp.

Abstract: Individual addition of a commercially available nutritional supplement and a methanol extract from an industrial Saccharomyces sp. strain SMC resulted in the enhanced growth of Saccharomyces sp. strain SMC in minimal medium. Isolation of the growth enhancing components from aqueous extracts of the supplement and the cellular extract was performed using reversed-phase, gel filtration, and ion exchange chromatography. Reversed-phase chromatography using Sep-Pak® vac C18 yielded aqueous washes which elicited increased yeast growth. Gel filtration chromatography of the aqueous washes in a group separation mode using Sephadex G25 gave three distinct groups for the nutritional supplement, and four distinct groups for the cellular extract. Fraction groups that exhibited growth enhancing activity also exhibited high absorbances at all three wavelengths of 214, 260, and 280 nm. Two major fractions which tested positive for growth enhancing activity in succeeding experiments were obtained after passing each of the active GFC groups through a Toyopearl SP 550C cation exchanger column. The active component from the cellular extract did not bind to the cation exchanger. The absorbance data at 214 nm (peptide bond experimental absorbance maximum wavelength), the Bradford assay (showing the presence of proteinaceous matter), and the active component’s inclusion in the Sephadex G25 fractionation range of 1-5 kDa (characteristic of small peptides) suggest that the growth enhancing components of the nutritional supplement and methanol cell extracts are peptides.