Method For Determination Of Protein Research Articles

The Anaphylactoid Constituents in Xue-Sai-Tong Injection

Xue-Sai-Tong injection, a traditional Chinese medicine with total saponins of Sanqi ginseng as active ingredients, has been used for more than 500 years to treat coronary artery disease in China. Anaphylactoid reaction induced by Xue-Sai-Tong injection was one of the main adverse drug reactions which has occurred frequently in recent years. It is of importance to elucidate its anaphylactoid constituents. The in vivo anaphyalctoid tests indicated that the anaphylactoid mediators could be used as indexes to evaluate the anaphylactoid action. Further, the in vitro model based on determining the mediators release from the degranulation of mast cells and RBL-2H3 cells stimulated by Xue-Sai-Tong injection was explored. Mediators released from mast cells and RBL-2H3 cells caused by Xue-Sai-Tong injection were determined by comparison of the methods of fluorospectrophotometry, ELISA, and spectrophotometry, respectively, revealing that the histamine release induced by the Xue-Sai-Tong injection could not be assayed accurately by the method of fluorospectrophotometry because of the interference of saponins and unknown components in the injection. The rat peritoneal mast cell was also not an optimal cell model for determining histamine and β-hexosaminidase release due to the higher spontaneous release ratio during the cell collection. Thus, ELISA determination of the histamine release from RBL-2H3 cells is a suitable in vitro model to assay the anaphylactoid reaction of Xue-Sai-Tong injection. Previously, abnormal hemolysis in some batches of Xue-Sai-Tong injection was observed in the course of their HD₅₀ (half hemolytic dosage) determination. This study further found that injections which exhibited an abnormal hemolysis phenomenon also caused a higher release of the anaphylactoid mediators from RBL-2H3 cells, indicating the HD₅₀ could be an auxiliary index to evaluate anaphylactoid action of the herbal injection indirectly. Research for anaphylactoid components in Xue-Sai-Tong injection indicated that proteins with over 10 KDa of molecular weight, but not ginsenosides, could be the main constituents inducing the release of anaphylactoid mediators from RBL-2H3 cells. An HPLC method for protein determination in the Xue-Sai-Tong injection was established subsequently, and the content of proteins with molecular weights of over 10 KDa in the injections showed an obviously positive correlation with the histamine release induced by the injections. In addition, taking ginsenoside-Rd coupled with BSA as an example, the hapten property of ginsenosides was studied and the ratio of ginsenoside-Rd to BSA was determined to be 8:1 by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and the ginsenoside-BSA conjugate showed a stronger action to stimulate histamine release from the RBL-2H3 cells.

Development of an ingenious method for determination of Dynabeads protein A based on a giant magnetoimpedance sensor

A novel analytical method based on giant magnetoimpedance (GMI) effect is developed for quantitative determination of Dynabeads protein A (DPA). An integrated GMI sensor involving the sensing elements of NiFe/Cu/NiFe films is prepared by Micro-Electro-Mechanical-Systems (MEMS) technology. DPA with various concentrations are immobilized on Au film, and a GMI-based biosensing platform is established. The magnetic signals produced by DPA are sensed by the GMI sensing elements, and the GMI sensor provides a detection signal in the form of a magnetoimpedance change. We observed that the GMI ratio is greatly reduced at high frequencies due to the presence of DPA. The GMI sensor displays a linear range of 0.1–5μgmL−1, and a low detection limit (90 Dynabeads) is achieved at f=1.5MHz. Therefore, this GMI-based analytical method may be useful for highly sensitive detection of biological markers in ultra-trace quantities.

Advanced Solid-State NMR Approaches for Structure Determination of Membrane Proteins and Amyloid Fibrils

Solid-state NMR (SSNMR) spectroscopy has become an important technique for studying the biophysics and structure biology of proteins. This technique is especially useful for insoluble membrane proteins and amyloid fibrils, which are essential for biological functions and are associated with human diseases. In the past few years, as major contributors to the rapidly advancing discipline of biological SSNMR, we have developed a family of methods for high-resolution structure determination of microcrystalline, fibrous, and membrane proteins. Key developments include order-of-magnitude improvements in sensitivity, resolution, instrument stability, and sample longevity under data collection conditions. These technical advances now enable us to apply new types of 3D and 4D experiments to collect atomic-resolution structural restraints in a site-resolved manner, such as vector angles, chemical shift tensors, and internuclear distances, throughout large proteins. In this Account, we present the technological advances in SSNMR approaches towards protein structure determination. We also describe the application of those methods for large membrane proteins and amyloid fibrils. Particularly, the SSNMR measurements of an integral membrane protein DsbB support the formation of a charge-transfer complex between DsbB and ubiquinone during the disulfide bond transfer pathways. The high-resolution structure of the DsbA-DsbB complex demonstrates that the joint calculation of X-ray and SSNMR restraints for membrane proteins with low-resolution crystal structure is generally applicable. The SSNMR investigations of α-synuclein fibrils from both wild type and familial mutants reveal that the structured regions of α-synuclein fibrils include the early-onset Parkinson's disease mutation sites. These results pave the way to understanding the mechanism of fibrillation in Parkinson's disease.

A novel water‐soluble polythiophene derivatives based fluorescence “turn‐on” method for protein determination

A lable‐free, simple, and sensitive fluorescence “turn‐on” approach is designed to rapidly detect protein using a conjugated polythiophene derivative (PDPMT‐Cl). The fluorescence of PDPMT‐Cl solution can be efficiently quenched by PtCl42− ions. Upon adding trypsin to the (bovine serum albumin, BSA) PDPMT‐Cl–PtCl42− solution, the BSA is cleaved into amino acid or peptide fragments, which are stronger PtCl42− ions chelators to form more stable complexes with PtCl42− ions. Thus, the PtCl42− ion is displaced from PDPMT‐Cl and the fluorescence of PDPMT‐Cl is recovered. By triggering the “turn‐on” signal of PDPMT‐Cl, it is successful to detect the protein in real time. “Turn‐on” response as readout signal is able to effectively reduce background noise and increase detection sensitivity. This method offers good selectivity for detecting protein in the presence of other common amino acids and metal ions. Under optimized conditions, the concentration of BSA in the range of 0.0004–1.75 mg/mL exhibits a linear relationship with the relative fluorescence intensity, and the correlation coefficient is 0.9997. The limit of detection is 4.47 × 10−4 mg/mL. The system is successfully applied for detecting protein in milk and egg. Due to the simplicity, sensitivity, and rapid response, this assay shows great potential for protein detection in the future. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 939‐943, 2013

Comparison of one-step and two-step methods for pI determination of proteins and polypeptides by capillary isoelectric focusing

One-step and two-step capillary isoelectric focusing (clEF) methods were employed the separation and pI determination of proteins and polypeptides. The parameters affecting the analysis efficiency, such as the sample solution, injection volume, focusing voltage, focusing time and driving conditions were optimized. The comparison of the two methods for separation of cytochrome C, hemoglobin, myoglobin, transferrin, bovine serum albumin and six polypeptides showed that the one-step cIEF was simple and fast, which could determine the pI of single component as well as it was rapid for protein and polypeptide separation, but it could not get good resolution or accurate pI of each component in a mixed sample. The two-step cIEF was more complex and needed longer time, however, which could separate and exactly determine the pI of each component in the mixture, and the pI value of each component determined was consistent with that determined using a single sample. The two methods are complementary, and can be widely used in rapid and accurate determination of the pI of amphiphilic biological particles.

Metal–tag labeling coupled with multiple reaction monitoring-mass spectrometry for absolute quantitation of proteins

Mass spectrometry-based quantitative proteomics, consisting of relative and absolute parts, has been used to discover and validate proteins with key functions related to physiological and pathological processes. Currently, stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) is the most commonly used method for the absolute determination of proteins in a biological sample. A prerequisite for this method is obtaining internal standards with isotope labels. Although many approaches have been developed for the labeling and preparation of internal peptides, expensive stable isotope labeling coupled with SID-MRM-MS has limited the application and development of an absolute quantitative method. Recently, a low-cost strategy using metal-tag labeling and MS has been developed for relative quantification of peptides or proteins. The introduction of labeling using metal tags has the merits of allowing multiple labeling and enlarging the mass shift to overcome the overlap of adjacent isotope clusters. However, most papers described MRM-MS for protein absolute quantification based on the metal in its peptides labelled with metal by inductively coupled plasma mass spectrometry (ICP MS) but not on its peptides labelled with metal. In this work, a novel approach based on metal-tag labeling coupled with MRM-MS was established for the absolute quantification of peptides or proteins. The principle of the method is that a bifunctional chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid bearing an N-hydroxysuccinimide ester (DOTA-NHS ester), is used to modify the N-termini of signature peptides from a target protein, and the modified peptides then chelate a certain metal, such as thulium, to form metal-tagged peptides (Tm-DOTA-P). Internal peptides are chemically synthesized and labeled with another metal, such as terbium (Tb-DOTA-P), as the internal standard. Both the Tb-DOTA- and Tm-DOTA-labeled peptides in samples can be analysed via MRM-MS. The experimental results show that the accuracy (%RE) and precision (%RSD) of the approach are both below 15%, and the lower limit of quantification (LOQ) is 0.8 fmol μL(-1), with good linearity (R(2) > 0.99) observed covering the range of 2 orders of magnitude. Furthermore, one protein, enolase, in an extract from Thermoanaerobacter tengcongensis was successfully quantified, which demonstrates that this novel absolute quantification method is a new strategy for simple, rapid, low-cost and accurate absolute protein quantification in a complex biological sample.

A simple rapid method for protein determination

A simple, rapid method is described for determination of protein in the range of 20-200 µg. The procedure is based on the reaction between protein and amido black 10B. An insoluble residue is precipitated and the dye concentration in solution decreases. The difference between the optical density of the blank (without protein) and sample with protein is a linear function of protein content.

Sensitive Method for Determination of Protein and Cell Concentrations Based on Competitive Adsorption to Nanoparticles and Time-Resolved Luminescence Resonance Energy Transfer between Labeled Proteins

A sensitive mix-and-measure method for the determination of protein and cell concentrations was developed. It is based on the competitive adsorption between the analyte and donor- and acceptor-labeled proteins to carboxylate-modified polystyrene nanoparticles. A high time-resolved luminescence resonance energy transfer (TR-LRET) signal is detected in the absence of the analyte due to the close proximity of the nanoparticle-adsorbed labeled proteins. The increased concentration of the analyte decreases the adsorption of the labeled proteins, leading to the loss of proximity and thus a decrease in the TR-LRET. The detection limit of the assay was 2.6 ng of proteins, which is higher than that of the most sensitive commercial methods. The method was also applied to cell counting, and 200 eukaryotic cells were measured in a microtiter well under the optimized conditions. The average coefficient of variation for both developed assays was approximately 10%, and the protein-to-protein variability for 11 different proteins was no more than 20%. The developed method requires no labeled particles, making the concept optimally applicable to varying targets as the material of the particle may be selected according to the application.

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

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/dm3. Calibration curves fulfilling second-degree polynomial equations can be determined for PDDA monomer units concentrations not exceeding 0.03 mmole/dm3.

Protein Quantitation Using Ru-NHS Ester Tagging and Isotope Dilution High-Pressure Liquid Chromatography–Inductively Coupled Plasma Mass Spectrometry Determination

An accurate, simple, and sensitive method for the direct determination of proteins by nonspecies specific isotope dilution and external calibration high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) is described. The labeling of myoglobin (17 kDa), transferrin (77 kDa), and thyroglobulin (670 kDa) proteins was accomplished in a single-step reaction with a commercially available bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis(hexafluorophosphate) (Ru-NHS ester). Using excess amounts of Ru-NHS ester compared to the protein concentration at optimized labeling conditions, constant ratios for Ru to proteins were obtained. Bioconjugate solutions containing both labeled and unlabeled proteins as well as excess Ru-NHS ester reagent were injected onto a size exclusion HPLC column for separation and ICPMS detection without any further treatment. A (99)Ru enriched spike was used for nonspecies specific ID calibration. The accuracy of the method was confirmed at various concentration levels. An average recovery of 100% ± 3% (1 standard deviation (SD), n = 9) was obtained with a typical precision of better than 5% RSD at 100 μg mL(-1) for nonspecies specific ID. Detection limits (3SD) of 1.6, 3.2, and 7.0 fmol estimated from three procedure blanks were obtained for myoglobin, transferrin, and thyroglobulin, respectively. These detection limits are suitable for the direct determination of intact proteins at trace levels. For simplicity, external calibration was also tested. Good linear correlation coefficients, 0.9901, 0.9921, and 0.9980 for myoglobin, transferrin, and thyroglobulin, respectively, were obtained. The measured concentrations of proteins in a solution were in good agreement with their volumetrically prepared values. To the best of our knowledge, this is the first application of nonspecies specific ID for the accurate and direct determination of proteins using a Ru-NHS ester labeling reagent.

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

Detection of carbonic anhydrase IX protein in the diagnosis of malignant pleural effusion by enzyme‐linked immunosorbent assay and immunocytochemistry

Carbonic anhydrase IX (CAIX) is a hallmark of metabolic change in cancer cells. The aim was to evaluate the utility of CAIX expression for the detection of malignant pleural effusions by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry. A total of 97 pleural effusions including 54 benign effusions, 10 atypical effusions, and 33 malignant effusions, classified based on cytological diagnosis and etiology, were subjected to ELISA to measure protein level and to immunocytochemistry in cell blocks to determine CAIX expression. CAIX levels were significantly higher in the malignant group compared with the benign group. Receiver operating characteristic curve analysis of benign and malignant pleural effusion for CAIX indicated an area under the curve of 0.82 with a value of 1882 pg/dL as the best threshold for distinguishing benign from malignant effusions, which yields a sensitivity, specificity, and accuracy of 63.6%, 94.4%, and 82.8%, respectively. None of the benign effusion expressed CAIX by immunocytochemistry. Malignant effusion cell blocks expressed CAIX in 63.6% of cases with 100% specificity. Upon combination of CAIX level and immunocytochemistry to detect malignant pleural effusions, the sensitivity and accuracy increased to 81.8% and 89.7%, respectively. The CAIX level determined by ELISA and the CAIX expression detected by immunocytochemistry were positively correlated. In atypical effusions, 5 cases (50%) exhibited CAIX levels higher than the cutoff value or were highlighted by CAIX immunocytochemistry. ELISA and immunocytochemistry methods for determination of CAIX protein are potential additional tests for detection of malignant pleural effusions.

Determination of main components in the extracellular polymeric substances extracted from activated sludge using a spectral probing method

In this study, a spectral probing method was applied to determine the content of the main components, i.e., proteins, polysaccharides and humic substances, in the extracellular polymeric substances (EPS) extracted from activated sludge. The measurement results were consistent with those obtained from the conventional methods, such as the anthrone for polysaccharide determination, the modified Lowry method for protein and humic substance determination. The recoveries for the determination of proteins, humic substances and polysaccharides in the EPS extracted from six sludge samples using standard additional method were between 92.4 and 108.9%, 84.8 and 108.9%, 75.1 and 117.2%, respectively. These results indicate that the propose method has a good accuracy and precision, and can be used as an effective approach to determine the main components in sludge EPS.

Protein quantitation using dyes obtained from plant materials

We present a preliminary report on the use of plant dyes in the quantitation of proteins in solution. We have used ethanol, acid, alkali and water to extract dyes from some plant materials, including flowers of Jungle flame (Izora coccinea), China rose (Hibiscus rosa-sinensis) and leaves of West African Indigo (Lonchocarpus cyanescens), Mimosa (Mimosa pudica), Roselle (Hibiscus sabdarifa), Jatropha (Jatropha curcas) and Henna (Lawsonia inermis). The dyes obtained were used in the protein-dye binding studies. The colour of the protein-dye complex of the ethanolic extracts was stable and increased linearly with increase in protein concentration. The extracts achieved linearity up to the following amount of proteins in the test samples: Hibiscus rosa-sinensis (60 mg), Ixora coccinea (120 mg), Hibiscus sabdarifa (80 - 100 mg), Jatropha curcas (80 mg), and Lawsonia inermis (100 mg). The sensitivity of the dyes especially at low protein concentrations indicate that they can provide suitable alternatives to other well known standard methods of protein determination.

Method for the Rapid Determination of Protein in Meats Using the CEM Sprint™ Protein Analyzer: Collaborative Study

Abstract A collaborative study was conducted to determine the protein content of raw and processed meat products by a protein-tagging and colorimetric technique. Meat products were prepared following AOAC Official MethodSM 983.18 and analyzed using CEM Corporation's Sprint Rapid Protein Analyzer. Sprint provides protein results by combining an accurately weighed test portion with a known amount of dye-binding agent. The dye-binding agent binds with the lysine, histidine, and arginine, as well as the n-terminus of the proteins commonly found in raw meat and processed meat products. Results are displayed and reported by the Sprint as a percentage (g/100 g) of protein. Ten blind duplicate study samples were sent to 10 collaborating laboratories in the United States. The within-laboratory (repeatability) relative standard deviation (RSDr) ranged from 0.91 to 3.04%, and between-laboratories (reproducibility) relative standard deviation (RSDR) ranged from 1.50 to 3.41% for protein. The method is recommended for Official First Action.

Resonance Light Scattering Method for Microdetermination of Proteins Based on Aggregation of Au Nanoparticles

By using a resonance light scattering (RLS) technique, a highly sensitive method for protein determination based on the aggregation of Au nanoparticles on protein template is described. For the Au nanoparticles of 15 nm, the detection limit of bovine serum albumin was 5.0 ng/mL and the linear range was 10–300 ng/mL. The experimental results indicated that various metal ions do not interfere with this assay. The proposed RLS assay exhibited lower variation in response signals for the same weight of different proteins and showed satisfactory results when it was used for determination of proteins in human serum.

Promoted negative staining of proteins in SDS‐PAGE using Eosin B compounded with magnesium chloride

In this study, we describe an effective visualizing technique for proteins in SDS-PAGE based on the organic dye, Eosin B, the sensitivity of which can be further strengthened by the addition of magnesium to the staining solution after electrophoresis. The newly developed protocol is low in cost and easily performed compared with the common methods for protein analysis in 1-D and 2-D gels. It provides a much better sensitivity (0.2 ng of single protein band) than that of imidazole-zinc negative stain for fixing and staining within 1 h, and an excellent performance in terms of compatibility with MALDI-TOF MS. The results show that similar identification scores and numbers of matched peptides were obtained by both methods. Furthermore, the effects of different metal salts on the quality of protein visualization by Eosin B were also investigated. Because of its sensitivity, stability, and safety, this stain may be a more practical method for protein determination in the routine laboratory.

Isolation and Purification of Jacalin from Artocarpus Heterophyllus Lam

This paper presents investigation results of saturation conditions needed for purification of jacalin lectin from the extract seeds of Artocarpus heterophyllus by ammonium precipitation and affinity chromatography on Galactose-Affi gel Hz. Three different aspects of parameters encompassing the percentage of saturation of ammonium sulfate precipitation, the presence of ammonium sulfate on Lowry method and the suitable galactose concentration for optimum elution of the protein from Galactose-Affi gel Hz were investigated. With three different sets of fractional saturation of jacalin purification using ammonium sulfate precipitation, the maximum yield of 0.463 g/g was achieved at 0-90% saturation range in the absence of dialysis. Maximum yield of 0.425 g/g was obtained at 30-60% and 0-90% saturation range in the presence of dialysis. The result from this work also indicates that excessive quantity of NH4SO4 interferes with Lowry method for protein determination substantially. The 0-90% saturation range was found to be more potentially appropriate for large scale application than 30-60% saturation, since the former involves only 1 step NH4SO4 addition. From the affinity chromatography, elution of 0.2 M galactose (in 0.15 M NaCl) from Galactose-Affi gel Hz produced the maximum peak profile and jacalin concentration. A reduction or increase in galactose concentration of more than 0.2 M did not increase concentration of purified jacalin purified using this method.

A novel capillary electrophoretic method for protein determination using bicinchoninic acid-Cu<SUP>+</SUP>colorimetric reaction

A new rapid, sensitive method for protein determination using capillary electrophoresis and specific colorimetric reaction of bicinchoninic acid (BCA) was established, assisted by microwave incubation. With 60 mmol/L boric acid buffer (pH 9.5) and inclusion additive of beta-cyclodextrin, the complex of BCA-Cu+ and free BCA molecules were efficiently separated. The peak intensity of BCA-Cu+ was higher than those of native proteins about two orders of magnitude at a low wavelength of 200 nm. The linear ranges of this method were from 2 to 200 mg/L for transferrin, and 2 to 100 mg/L for ricin. The limits of detection for transferrin and ricin were 0.33 and 0.37 mg/L, respectively. This method was also successfully applied in the determination of some ricin samples in the First International Proficiency Test on the quantification of ricin.

Technical note: Comparing calibration methods for determination of protein in goat milk by ultraviolet spectroscopy

A rapid spectroscopic method to determine total protein in bovine and buffalo milk using UV spectra of guanidine-hydrochloride mixed milk has previously been reported and validated. The method was based on mixed calibration samples and univariate calibrations of fourth derivative (4D) spectra. In this study the same method was compared and tested for determination of total protein in goat milk. Calculations based on multivariate calibration (partial least squares regression) on full spectra of goat milk were used. The method was tested on 2 UV instruments. The comparison resulted in a significantly more robust (i.e., better) transferability between UV instruments for the partial least squares regression method on full spectra compared with previous univariate calibration of 4D spectra. Local (1 instrument) calibrations gave similar, significantly not different (chi-squared test) cross-validated prediction error results for the 2 methods. It can be concluded that there is no need for fourth derivation. Partial least squares regression on full spectra was equal or superior to using the 4D spectra.