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Gingival mesenchymal stem cell-conditioned medium (GMSC-CM) is a conditioned medium obtained from cultured gingival mesenchymal stem cells . GMSCs are easily isolated from gingival tissue, whereas gingival tissue is easily obtained by minimally invasive techniques. On the other hand, C M contains proteins, cytokines, chemokine s and growth factors that play an important role in osteogenic differentiation. This study aim s to determine the total protein and calcium levels in GMSC-CM. GMSCs were grown in culture media with 10% of FBS. CM of GM SC was obtained from the media collection process with a 0.22 µm filter and concentrated with a centrifugal filter to obtain a concentrated GMSC-CM. The concentration of total protein was performed by bicinchoninic acid protein assay on GMSC-CM and concentrated GMSC-CM. Calcium level was performed by atomic absorption spectroscopy method. The results are that GMSC-CM had a total protein concentration of 2502±0.06 µg/ml, and the concentrated GMSC-CM was 1.912±0.08 μg/ml. The calcium level of GMSC-CM was 0.0 09 % and concentrated GMSC-CM was 0.0 09 %. It can be concluded that GMSC-CM had a high concentration of total protein and calcium. These parameters were used to develop bioprocesses to enhance the production of GMSC-CM, which will support the implementation of cell-free therapy for tissue regeneration.

Context. Proteins in the saliva are one of the defense mechanism factors that can protect the oral cavity from disease. However, smoking might affect the properties of saliva. Aim To determine the differences in salivary protein profiles and total concentrations in smokers and non-smokers and their correlation with dental caries severity as indicated by the Decayed, Missing, Filled-Teeth (DMF-T) scores. Methods and material This cross-sectional study included 25 smokers and 25 non-smokers. The DMF-T scores were recorded. The total salivary protein was measured by the Bradford method, and the profile proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results The average of salivary protein concentration in smokers was lower than that in non-smokers (551.486 µg/mL versus 765.361 µg/mL), but the difference was not statistically significant (P > 0.05). Further correlation analyses showed a negative correlation between the concentration of proteins based on the extent of smoking. A weak negative correlation was found between protein concentration and DMF-T scores (r = −0.239). Dominant salivary protein bands of 11.6 kDa and 54.5 kDa were found in smokers and 27 kDa, 60 kDa, and 94.5 kDa were found in non-smokers. Conclusion Different protein bands appeared in smokers and non-smokers. There was a weak correlation between protein concentration, DMF-T scores, and the extent of smoking.

The aim of our work was to develop a low-cost, portable device for the fast and easy determination of total protein content by using PDMS-based lab-in-a-syringe technology with removal of 3D-printed channels. We proposed two designs with a one-step PDMS curing and a two-step PDMS-curing fabrication procedure. The one-step PDMS microdevices were found to be the best in the view of preparation, repeatability, and stability of the reagent. This design was then applied for the determination of total protein content in biomedical products using the Bradford assay.

Objectives: This study aimed to obtain an optimum method for zincyeast production. Materials and Methods: Production of zinc-yeast was prepared using Yeast Extract Peptone Dextrose (YEPD) as a culture medium. Yeast that had been inoculated into culture media was incubated with a shaker at room temperature for 84 hrs, then zinc sulfate was added with various concentrations of 200, 300, and 400 mg/L, and then incubation continued for 24 hrs. The obtain yeast biomass was dried with a Freeze-dryer and analyzed for total accumulated zinc and protein content. Determination of zinc was using atomic absorption spectrophotometry (AAS) at a wavelength of 196 nm, whereas protein content was determined with Bradford method at a wavelength of 595 nm using UV-Vis spectrophotometer. Results: Zinc-yeast with the addition of 200 mg/L zinc sulfate obtained 0.3111 g that contains 2228.60 μg/g of zinc and 0.8515 mg/L of protein. While zinc-yeast with the addition of 300 mg/L zinc sulfate obtained 0.3704 g that contains 2458.30 μg/g of zinc and 0.8682 mg/L of protein. And the last, zinc-yeast with the addition of 400 mg/L zinc sulfate obtained 0.2370 g that contains 2648.43 μg/g of zinc and 0.8935 mg/L of protein. Conclusion: Production of zinc-yeast with the highest zinc and protein content can be obtained by adding 400 mg/L zinc sulfate solution to the stationary phase culture of S. cerevisiae. The modified zinc-yeast production method is simple and can be used for the production of functional food products.

Objectives: The objective of this study was to perform selenium yeast production and analyze the selenium and protein contained in selenium yeast obtained. Methods: Selenium yeast production was carried out by incubating the stationary phase culture of yeast with selenium solution for 24 hr, and yeast biomass was dried by Freeze-dryer to produce selenium yeast in powder form, then the selenium yeast was determined to the selenium content with atomic absorption spectrophotometer at a wavelength of 196 nm and protein content analyzing by the Bradford method at a wavelength of 595 nm using UV-Vis spectrophotometer. Results: Selenium yeast production reached 2.5 g of selenium yeast in powder form. The selenium and protein content of selenium yeast was 4258.0096 (±278.39) μg Se/g and 0.8505 (±0.0045) mg/mL, respectively. The method was easy, simple, and can prove that selenium yeast can still be produced and analyzed for the selenium and protein content. Conclusion: The present study demonstrated that production of selenium yeast in the form of dry powder could be carried out using the stationary phase culture of S. cerevisiae incubated with 30 μg/mL selenium solution, and the resulting selenium yeast can be analyzed for selenium content using atomic absorption spectrophotometer and protein content by Bradford method using UV-Vis spectrophotometer.

In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.Keywords: recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification

The fabrication of sensitive protein microarrays such as PCR used in DNA microarray is challenging due to lack of signal amplification. The development of microarrays is utilized to improve the sensitivity and limitations of detection towards primal cancer detection. The sensitivity is enhanced by the use of ZnO-nanorods and is investigated as a substrate which enhance the florescent signal to diagnose the hepatocellular carcinoma (HCC) at early stages. The substrate for deposition of ZnO-nanorods is prepared by the conventional chemical bath deposition method. The resultant highly dense ZnO-nanorods enhance the fluorescent signal 7.2 times as compared to the substrate without ZnO-nanorods. The microarray showed sensitivity of 1504.7 ng ml-1 and limit of detection of 0.1 pg ml-1 in wide dynamic range of 0.05 pg-10 μg ml-1 for alpha fetoprotein (AFP) detection in 10% human serum. This immunoassay was successfully applied for human serum samples to detect tumor marker with good recoveries. The ZnO-nanorod substrate is a simple protein microarray which showed a great promise for developing a low-cost, sensitive, and high-throughput protein assay platform for several applications in both fundamental research and clinical diagnosis.

Objectives: To optimize an enzyme-linked immunosorbent assay (ELISA) for measuring the HspX protein (α-crystallin) levels and then evaluate its correlation with the accumulation of lipid bodies in Mycobacterium bovis (M. bovis) during hypoxia and exposure to nitric oxide. Methods: This study was conducted at Prince Sultan Military Medical City, Riyadh, Saudi Arabia between 2016 and 2017. We first optimized ELISA conditions for the detection of HspX. The optimization protocol focused on minimizing concentrations of the capture antibody, detection antibody, and conjugated secondary antibody, and determining the minimum detection limit of the antigen, HspX. Bacteria were grown either in shaking culture or in stationary flasks mimicking hypoxic environments. A standard Bradford assay was used to determine the total protein and HspX was detected using the optimized ELISA protocol. The effect of hypoxic environment and nitric oxide on the levels of HspX and lipid bodies, detected by staining with Nile red, was also evaluated. Results: An optimized ELISA protocol was established for the detection of HspX from M. bovis. Exposure to nitric oxide and hypoxic conditions led to an increase in the levels of HspX protein. The increase in HspX associated with nitric oxide treatment and hypoxic conditions correlated with higher levels of lipid bodies mainly found in pathogenic mycobacteria. Conclusions: The optimized ELISA protocol in this study can detect HspX protein levels in M. bovis growing in normal and hypoxic environments. Importantly, hypoxia led to enhanced expression of HspX protein, which correlated with the enhanced production of lipid bodies. Lipid body production is a survival strategy of pathogenic mycobacteria.

Past investigations involving fingerprints have revolved heavily around the image of the fingerprint-including the minutiae, scarring, and other distinguishing features-to visually find a match to its originator. Recently, it has been proven that the biochemical composition can be used to determine originator attributes, such as sex, via chemical and enzymatic cascades. While this provides pertinent information about the originator's identity, it is not the only piece of information that can be provided. This research was designed with three goals in mind: (1) identify how long it would take before an aged female fingerprint could no longer be differentiated from a male fingerprint, (2) identify a correlation between the data collected and a specific time since deposition (TSD) time point, and (3) identify if a specific amino acid could be contributing to the decreasing response seen for the aging fingerprints. Using ultraviolet-visible (UV-vis) spectroscopy, aged fingerprints were evaluated over the course of 12 weeks via three chemical assays previously used for fingerprint analysis-the ninhydrin assay, the Bradford assay, and the Sakaguchi assay. As fingerprints age, the conditions they are exposed to cause the biochemical composition to decompose. As this occurs, there is less available to be detected by analytical means. This results in a less intense color production and, thus, a lower measured absorbance. The results displayed here afforded the ability to conclude that all three goals set forth for this research were accomplished-a female fingerprint can be differentiated from a male fingerprint for at least 12 weeks, UV-vis data collected from aged fingerprints can be correlated to a TSD range but not necessarily a specific time point, and the decomposition of at least a single amino acid can afford the ability to estimate the TSD of the fingerprint.

Introduction: Further development of magnetic-based detection techniques could be of significant use in increasing the sensitivity of detection and quantification of hepatitis B virus (HBV) infection. The present work addresses the fabrication and characterization of a new bio-nano composite based on the immobilization of goat anti-HBsAg antibody on modified core-shell magnetic nanoparticles (NPs) by (3-aminopropyl) triethoxysilane (APTES), named Fe3O4@SiO2/NH2, and magnetic NPs modified by chitosan (Fe3O4@CS). Methods: At the first step, Fe3O4 was modified with the silica and APTES (Fe3O4@SiO2/NH2) and chitosan (Fe3O4@CS) separately. The goat anti-HBsAg antibody was activated by two different protocols: Sodium periodate and EDC-NHS. Then the resulted composites were conjugated with activated goat anti-HBsAg IgG. An external magnet collected Bio-super magnetic NPs (BSMNPs) and the remained solution was analyzed by the Bradford method to check the amount of attached antibody to the surface of BSMNPs. Results: The findings indicated that activation of antibodies by sodium periodate method 15-17 µg antibody immobilized on 1 mg of super magnetic nanoparticles (SMNPs). However, in the EDC-NHS method, 8-10 µg of antibody was conjugated with 1 mg of SMNPs. The resulting bio-magnetic NPs were applied for interaction with the HBsAg target using enzyme-linked immunosorbent assay (ELISA). About 1 µg antigen attached to 1 mg SMNPs, which demonstrated that the fabricated materials are applicable in the detection scope of HBsAg. Conclusion: In the present study, we developed new antibody-conjugated magnetic NPs for the detection of HBsAg using an efficient conjugation strategy. The results demonstrated that the binding capacity of Fe3O4@SiO2/NH2 was comparable with commercially available products. Our designed method for conjugating anti-HBsAg antibody to a magnetic nanoparticle opens the way to produce a high capacity of magnetic NPs.

Lemna sp. is known to have several bioactive compounds and polysaccharide macromolecules that can function as immunomodulators to affect non-specific immune responses to increase the body's resistance to pathogens. This study aims to determine the potential of catfish eye extract as an immunostimulant by observing non-specific tilapia immune parameters. The extraction method used was 96% ethanol maceration for 2 days with a ratio of 1: 4. The experimental design used a Completely Randomized Design with 5 treatments (doses 0.2, 0.4, 0.6 mg/kg, control + and control -) and 3 replications. The non-specific parameters of immunity observed included total plasma protein (Bradford method), superoxide dismutase and lysozyme activity. The data obtained will be analyzed using ANOVA, if there is a significant difference, it will be further tested with Duncan Multiple. Range Test (DMRT). The results showed that the highest total plasma protein was found in treatment C (giving an extract of 0.3 mg/kg body weight) with an average total plasma protein after 12 days of maintenance of 4.99 g / dL. The extract dose of 0.3 mg/body weight showed a rapid decrease in SOD and increase Lysozyme activity.

Malaria is still an essential epidemiological disease worldwide, including in Indonesia. Several approaches are performed to control the disease, as well as vaccine development. The Cysteine-rich interdomain region α of Plasmodium falciparum erythrocyte membrane protein 1 (CIDRα-PfEMP1) is a pivotal domain in the malaria pathogenesis make it a malaria vaccine candidate. The development of the malaria vaccine is performed using recombinant technology. Recombinant protein production is an important step. The study aimed to determine the optimized condition for CIDRα-PfEMP1 recombinant protein expression in Escherichia coli BL21(DE3) expression system. Serial IPTG concentrations from 0.05, 0.1, 0.3, and 0.5 mM and two different incubation periods of 4 h and 8 h were optimized. The recombinant protein expression was visualized in SDS-PAGE, measured using the Bradford protein assay, and calculated using software Image J. SDS-PAGE visualization showed a 27 kDa band expressed CIDRα-PfEMP1 recombinant protein. The optimized condition for CIDRα-PfEMP1 recombinant protein expression was at 0.03 mM IPTG concentration and 8 h incubation period.

Yifan Wang,1– 3 Si Chen,1– 3 Xin Yang,1– 3 Shuang Zhang,1– 3 Chunying Cui1– 3 1Department of Pharmaceutics, School of Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China; 2Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing, People’s Republic of China; 3Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Beijing, People’s Republic of ChinaCorrespondence: Chunying Cui; Shuang ZhangDepartment of Pharmaceutics, School of Pharmaceutical Science, Capital Medical University, Number 10 Youanmenwai Street, Fengtai, Beijing, 100069, People’s Republic of ChinaTel +86-10-8391-1668; +86-10-8391-1673Fax +86-10-8391-1668; +86-10-8391-1673Email ccy@ccmu.edu.cn; zshuang@ccmu.edu.cnBackground: siRNA brings hope for cancer therapy. However, there are many obstacles for application of siRNA in clinical. Because of the excellent biocompatibility, non-toxicity and non-immunogenicity of bovine serum albumin (BSA), BSA-based nanoparticles have been widely designed as a drug carrier system.Methods: The optimal formula for BSA NPs preparation was investigated by central composite design response surface methodology (CCD-RSM), BSA-based survivin-siRNA delivery system (BSA NPs/siRNA) was characterized by dynamic light scattering, atomic force microscope, transmission electron microscope and Bradford method. The in vitro anti-tumor effect and mechanism of BSA NPs were investigated by confocal microscopic imaging, MTT assay, RT-qPCR and ELISA analysis. Moreover, the anti-tumor effect, distribution and biosafety of BSA NPs were studied in vivo.Results: The optimal formula for BSA NPs was settled to be 20 mg/mL for BSA concentration, 9 for pH value, 136% for crosslinking degree and 1.6 mL/min for speed of ethanol addition. BSA NPs/siRNA could remain stable at 4°C for 4 weeks and protect siRNA from degradation by RNase A. Besides, BSA NPs/siRNA could maintain a sustained release of siRNA and promote the uptake of siRNA significantly. The survivin-mRNA level and the survivin-protein level were decreased by 55% ± 1.6% and 54% ± 1.6% separately. The in vivo tumor inhibition results suggested that the tumor inhibition rate of BSA NPs/siRNA-treated group was 54% ± 12% and was similar with that of DOX-treated group (57% ± 9.2%, P > 0.05). The biosafety results confirmed that BSA NPs/siRNA could not induce significant damages to the main organs and blood in vivo.Conclusion: These results demonstrated that CCD-RSM was an effective tool for preparation analysis, and the BSA NPs/siRNA was a promising system for siRNA-based gene therapy.Keywords: bovine serum albumin, BSA, CCD-RSM, survivin-siRNA, nanoparticles, RNAi

The results of modelling the effect of cell sizes on the microorganism disintegration process are presented. Experiments were conducted in a horizontal bead mill for baker's yeast Saccharomyces cerevisiae. The kinetics of the cell disruption process was investigated by optical image analysis and the release of intracellular protein by Bradford method. A large variation in the size of yeast from one production cycle has been demonstrated. A significant increase in the constant rate of the process with an increase in the range of the cell size class was demonstrated. More beneficial values of the coefficient of determination were obtained for the spherical model of the cell. Worse results of the process description were obtained for the ellipsoidal model. They may result from large variations in cell volume in individual size classes. The greatest changes in the cell Elongation parameter within one size class ranged from 1.00 to 1.54.

The development of an effective and noninvasive early method for obtaining fetal cells is crucial to prenatal screening. Despite proving the presence of fetal cells in the reproductive tract, their use is limited due to their inability to properly isolate them from maternal cells. Magnetic-activated cell sorting (MACS) is a simple technique to separate cells. The present study aimed to develop a MACS-based platform for the isolation of the HLA-G expressing trophoblast cells. For this purpose, first, the triazine functionalized MNPs were synthesized and characterized. Then, MNPs were directly and indirectly conjugated by the MEM-G/9 antibodies targeting HLA-G+ cells. The antibody amount on the surface of the nanoparticles was determined with the Bradford assay. The cell capture efficiency was also investigated. Various characterization methods confirmed the successful nanoparticle synthesis and antibody conjugation. The optimal initial antibody amount for the immobilization was about 20 μg and the optimal time was 3 h. The antibody-nanoparticles by the indirect method had better targeting and capture efficiency than the direct method. The MNPs indirectly conjugated with antibodies are an efficient tool for cell isolation and present considerable potential to be applied in biomedical fields.

Background. Keratins are natural biopolymers with a wide range of applications in the field of biotechnology. Materials and Methods. Extraction of keratins was performed by a modified Nakamura method using 250 mM DTT. The protein concentration in the supernatant was determined by Bradford method. The protein composition was studied by their electro­phoretic separation in a polyacrylamide gel in the presence of sodium dodecyl sulfate. The films were made by casting. The surface characteristics of the films were determined using a scanning electron microscope REMMA-102. The elemental composition of the films was determined using an X-ray microanalyzer. Results . The protein concentration in the supernatant was 3.75 mg/mL. After using dithiothreitol in the extraction mixture, we obtained proteins of intermediate filaments with a molecular weight of 40–60 kDa and a low Sulfur content. In the low molecular weight region, we obtained keratin-associated proteins with a molecular weight of 10–30 kDa and a high content of Sulfur. These proteins belong to fibrillar proteins, which can be used as a matrix for the creation of new keratin-containing biocomposites with a wide range of applications in reparative medicine and tissue engineering. Based on the obtained keratin extract, polymer films with and without the addition of glycerol were made. Scanning electron microscopy revealed that glycerol provided the film structure with homogeneity and plasticity due to the accumulation of moisture after the fixation by water vapor. The X-ray microanalysis of films revealed such elements as Sodium, Silicon, Sulfur, Potassium. Among the detected elements, Sulfur has the largest share that is due to the large number of disulfide bonds in the keratin molecule. Conclusions. The polymer keratin films with the addition of glycerol demonstrated better mechanical properties and can be used in biomedicine. Keywords: keratins, hair, extraction, biopolymers Received 27 January, 2021 ● Revision 07 February, 2021 ● Accepted 17 March, 2021 ● Published 31 March, 2021

Lectins arecarbohydrate-bindingproteinsthat are highly specific for sugarmoietiesof other molecules. They perform recognition on the cellular and molecular level and play numerous roles in biological recognition phenomena involving cells, carbohydrates, and proteins. Blood groups are inherited characters which give rise to antigen-antibody reaction. A total of 120 samples of local (Nigeria)Achatina achatinasnail specie were collected, authenticated at the Zoology Department of the University of Nigeria, Nsukka and 80mls of pooled crude lectin extract was obtained. Purifications were performed on 20mls of the crude extract in three steps viz, Ammonium sulphate precipitation and Dialysis (Partial purifications), Con A Sepharose 4B affinity chromatography column (Complete purification). The affinity purified lectin was used for all the actual tests conducted in this research. The crude, partially and complete/affinity purified lectin extracts were subjected to Haemagglutination tests, Protein Assay and Specific Sugar determinations. The molecular weight was assessed by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The results of the research showed as follows: On complete/affinity purification, 15mls of pure sample containing only the high molecular weight lectin was obtained. The respective haemagglutination tests on the crude, partially and affinity purified lectin showed on standardisation, preferential agglutination with Blood group A type. The Protein contents of the lectin was deduced to be as follows: The crude extract contains 13.5mg/dl, Dialysed precipitate – 5.7mg/dl, Dialysed supernatant – 5.0mg/dl and the Affinity purified Lectin – 0.422mg/dl. Galactose N-acetyl amine (Gal NAc) residue was determined to be its specific sugar. The SDS-PAGE analysis showed the molecular weight of the lectin to be 250 KDaltons. This research has therefore succeeded in the Purification, Characterisation and illustration of the lectinic properties of the local Nigeria snail -Achatina achatina.

Protein extraction is a preliminary step of protein purification which mainly focus on maximization of total protein yield. The heterogeneous properties cause diversification of protein; therefore, there is no absolute protocol in protein extraction. The ratio of buffer gives different protein concentrations in different types of mammalian tissues, and this condition leads to the study of optimization of buffer ratio to obtain a better total protein yield. The objectives of this study were to compare the total protein yield based on three different ratios of buffer used. The phosphate buffer saline (PBS), radioimmunoprecipitation assay (RIPA) buffer, and RIPA buffer with the addition of protease inhibitor (Pi) were used with the ratios of 1:1, 1:3, and 1:5. Fetal cotyledons removed from the placenta have undergone mechanical disruption, incubation, sonication, and centrifugation. The supernatant was retained and quantified with Bradford assay to determine the total protein yield based on the standard curve of bovine serum albumin (BSA). There was a statistically significant different between buffer ratio (p

Milk bioactive peptides are derivative of milk protein produced either through enzymatic activity, digestive processes, or fermentation, that give functional properties. The study aimed to obtain bioactive peptides fraction derived from goat’s milk casein through hydrolysis by papain, analyze the profiles of protein and peptides, and also test the antioxidative activity. The casein isolate was hydrolyzed by papain in a ratio of 100: 0.5 (v/v) at pH 7.0 and 50ºC. Analysis of protein concentration was carried out by the Bradford method and protein profile by SDS-PAGE. Antioxidant assay was carried out by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. A 1,000 ppm ascorbic acid solution was used as positive control. Peptide fractionation was done by membrane filtration with a cut off of 10 kD and 30 kDa. The protein concentration of casein hydrolysates decreased significantly after hydrolysis process with papain. The electrophoresis results showed six protein bands in casein with molecular weight of 7-33 kDa. After the hydrolysis process, all hydrolysates only contained two protein bands with molecular weights of 8 and 5 kDa. The hydrolysis process increased the antioxidant activity of the casein. P0 and P2 hydrolysates had the highest antioxidant activity, and fractions with the highest antioxidant activity were fraction

Artificial Insemination Center (AIC) produces frozen semen that was taken from superior bull. The selection of superior bull was based on breeding soundness evaluation. This study is aimed to evaluating the correlation between the molecular weight (MW) of seminal plasma protein and semen quality of Simmental bulls. Semen ejaculates from nine Simmental Bulls, that belong to AIC of Central Java, at Ungaran, were collected by using an artificial vagina. The sub population of sperm in ejaculates was evaluated through macroscopically and microscopically, in which the semen then centrifugated 6500 rpm for 30 minutes. The supernatant was collected and inserted into mini straw and stored in liquid nitrogen container. The concentration of seminal plasma protein was determined by the Bradford method. The Bradford protocol of analysis was suitable with User Guide Coomasize (Bradford) Protein Assay Kit. Thermo Skanlt RE for Multiskan Go Software, 3.2 version was applied for analyzing of the data, and protein characterization used was 1D-SDS-PAGE. The Coomassie Brilliant Blue was used for coloring the gels, and the proteins MW was determined by MW markers. The result demonstrated that there was no significant correlation between protein concentration and semen quality (P>0.05). Protein expressions based on MW were 62-48 kD, 100-71 kD found in this study. The analysis of correlation showed that the proteins with 100-71 kD MW correlated with sperm motility, normal sperm morphology, and sperm concentration. Semen quality in all parameters was significant (P˂0.05 and P>0.01) with 62-48 kD protein MW. The result concluded that seminal plasma protein had a correlation with semen quality and can be used as additional indicator for bull’s candidate selection.

Introduction Protein corona (PC) deposition on nanoparticles (NPs) in biological systems contributes to a great extent to NPs' fates; their targeting potential, the interaction with different biological systems and the subsequent functions. PC - when properly tuned - can serve as a potential avenue for optimization of NPs' use in cancer therapy. Methods Poly-lactic co-glycolic acid (PLGA)-based NPs exhibiting different physicochemical properties were fabricated and characterized. The PC makeup of these NPs were qualitatively and quantitatively analyzed by Western blot and Bradford assay, respectively. The effect of PC on the release of NPs' cargos and the intracellular uptake into B16F10 melanoma cells has been studied. Results The composition of NPs (polymeric PLGA NPs vs lipid-polymer hybrid NPs) and the conjugation of an active targeting ligand (cRGDyk peptide) represented the major determinants of the PC makeup of NPs. The in vitro release of the loaded cargos from the NPs depended on the PC and the presence of serum proteins in the release medium. Higher cumulative release has been recorded in the presence of proteins in the case of peptide conjugated NPs, cNPs, while the unconjugated formulations, uNPs, showed an opposite pattern. NPs intracellular uptake studies revealed important roles of distinct serum and cellular proteins on the extent of NPs' accumulation in melanoma cells. For example, the abundance of vitronectin (VN) protein from serum has been positively related to the intracellular accumulation of the NPs. Conclusion Careful engineering of nanocarriers can modulate the recruitment of some proteins suggesting a potential use for achieving endogenous targeting to overcome the current limitations of targeted delivery of chemotherapeutic agents.

The present study aims to investigate the effects of exposure of the seminal plasma and spermatozoa of the giant grouper Epinephelus lanceolatus to dry ice (−79 °C) during transport on their quality. In all, 15 amino acid compounds were determined. The quantification of total proteins were measured using the Bradford method, and amino acid concentration were measured using the HPLC method. The cryopreserved seminal plasma was transferred from a liquid nitrogen tank to a styrofoam box filled with dry ice. Total protein and amino acids were measured after 24, 48, and 72 h. For comparative purposes, total protein and fifteen compound of amino acid were also measured. Both parameters were also measured after the cryopreserved seminal plasma were immersed in liquid nitrogen after 24 and 48 h exposed to dry ice. The results showed that the exposure of seminal plasma to dry ice for 24, 48 and 72 h during transportation or immersion back into the liquid nitrogen after 24 and 48 h does not change the total protein levels either in seminal plasma or spermatozoa. However, the level of each amino acid compound in the seminal plasma had significantly decreased.

Abstract Characterization of selenium nanoparticles and selenium nanoparticle–human serum albumin conjugates prepared in nanopowder form, their elemental composition, and particle size distribution were investigated with a microwave plasma optical emission spectrometry operating in a single-particle mode. This new analytical technique was used for the first time to examine the molecular interaction between selenium nanoparticles and human serum albumin regarding potential biomedical applications of selenium nanoparticles. Nanopowder sample was introduced to a helium plasma by pneumatic nebulization based on fluidized bed approach and measured with a time resolution of 20 ms. Both selenium nanoparticles and selenium nanoparticle–human serum albumin conjugates were characterized by observation of synchronous signals from different particle components. Plots of the time correlation between Se and C signals for all particles in selenium nanoparticles and selenium nanoparticle–human serum albumin conjugates samples differed from each other in degree of correlation and synchronicity of recorded signals. The interaction between selenium nanoparticles and human serum albumin was confirmed using Bradford assay. For selenium nanoparticles synthesized using yeast cells, the percentage of bound protein was only of 4%, whereas for selenium nanoparticles synthesized using yeast extract as a stabilizing agent it was 16%. Graphic abstract

The objective of this study was to correlate the Biuret, Lowry, Bradford, and Mlikoscan methods used to determine the level of protein in goat's milk and compare them with the Kjeldahl method. Milk samples collected from 18 goats of breeds Saanen (n=5), Anglo-Nubian (n=4), Pardo-alpina (n=5), and Creole (n=4) were analyzed. Correlation analyses between the different methods were performed using the Pearson test and the protein content between different breeds was compared using ANOVA. Total protein (TP) and casein (CN) concentrations obtained by all methods studied were positively correlated with the Kjeldahl method (p

Allergen protein is a protein that could triggers allergy reaction. This study purposes to examine the characteristic of allergen protein of unripe, boiled, and fried nila baby fish. Characteristic of allergen protein were observed by electrophoresis and immunoblotting method, while the determination of protein concentration was observed by Bradford method. Identification of the used nila baby fish was accomplished in Faculty of Fishery and Marine Science of IPB. The result of identification showed that the used nila baby fish was Oreochromis niloticus. The proximate analysis of unripe, boiled, and fried nila baby fish resulted water content ranged from 19.16%-23.68%, fat content ranged from 1.03%-20.44% and carbohydrate content ranged from 0.16%-20.27%. Protein concentration of unripe, boiled, and fried nila baby fish extract respectively were 1963.45 mg/L; 699.82 mg/L; 607.79 mg/L. The band amount of allergen protein of unripe, boiled, and fried nila baby fish which was detected by electrophoresis, respectively were 16 of protein band, 26 of protein band and 16 of protein band. The immunoblotting showed that the sum of respondent who contained specific IgE that can bind with allergen protein of boiled and fried nila baby fish were more than allergen protein of unripe nila baby fish. It indicated that the processing process by boiling and frying would increase allergenicity toward nila baby fish.

Antibiotic resistance against present antibiotics is rising at an alarming rate with need for discovery of advanced methods to treat infections caused by resistant pathogens. Silver nanoparticles are known to exhibit satisfactory antibacterial and antibiofilm activity against different pathogens. In the present study, the AgNPs were synthesized chemically and characterized by UV-Visible spectroscopy, scanning electron microscopy, and X-ray diffraction. Antibacterial activity against MDR K. pneumoniae strains was evaluated by agar diffusion and broth microdilution assay. Cellular protein leakage was determined by the Bradford assay. The effect of AgNPs on production on extracellular polymeric substances was evaluated. Biofilm formation was assessed by tube method qualitatively and quantitatively by the microtiter plate assay. The cytotoxic potential of AgNPs on HeLa cell lines was also determined. AgNPs exhibited an MIC of 62.5 and 125 μg/ml, while their MBC is 250 and 500 μg/ml. The production of extracellular polymeric substance decreased after AgNP treatment while cellular protein leakage increased due to higher rates of cellular membrane disruption by AgNPs. The percentage biofilm inhibition was evaluated to be 64% for K. pneumoniae strain MF953600 and 86% for MF953599 at AgNP concentration of 100 μg/ml. AgNPs were evaluated to be minimally cytotoxic and safe at concentrations of 15-120 μg/ml. The data evaluated by this study provided evidence of AgNPs being safe antibacterial and antibiofilm compounds against MDR K. pneumoniae.

Rice that is generally consumed by Indonesian people is that does not have important nutrient such as β-carotene. Therefore, the way to supply β-carotene requirement in staple foods can be done through β-carotene biofortification by genetic engineering of the rice plant. PAC Nagdong/ IR36 is transgenic Golden Rice which can produce β-carotene. The research aimed to determine some nutritional content of PAC Nagdong/ IR36 rice lines that include carbohydrate, amylose, amylopectin, protein, lipid, and β-carotene content. Amylose was measured using the iodine colorimetric method, whereas the contents of amylopectin and carbohydrates were measured using the by difference method. Protein was measured using the Bradford method, lipid was measured using Proctor and Bowen method, while β-carotene content was measured using the colorimetric method. PAC Nagdong/ IR36 rice lines had carbohydrate content between 85,06-90,16%, amylose 12,94%- 20,81%, amylopectin 69,35- 76,17%, protein 7,76-8,52 µg/µl, lipid 0,65-0,90%, and β-carotene 231,61-920,52 µg/g. Carbohydrate, protein, and β-carotene in PAC Nagdong/ IR36 were higher than TN1 rice variety (control). This indicated that PAC Nagdong/ IR36 provided better nutrition. Amylose content of Nagdong/ IR36 PAC rice was low, which donated its potential to produce fluffier rice whereas high β-carotene content could be used as a source of β-carotene that is useful in overcoming vitamin A deficiency and beneficial for health including in preventing degenerative diseases.

Human epidermal growth factor receptor 2 (HER2) status in breast cancer is routinely determined through immunohistochemistry (IHC) and/or in situ hybridisation (ISH) performed on whole tissue sections (WS). The purpose was to evaluate whether a gene protein assay (GPA) combining IHC with ISH, performed on breast cancer tissue microarray (TMA), is suitable for large-scale retrospective HER2 status evaluation. TMAs from 606 tumours from a Swedish population-based cohort (2005–2012) were stained with GPA. GPA IHC on TMA yielded weaker staining than IHC on WS during routine pathological assessment (86.0% agreement). However, final HER2 status agreement between GPA on TMA and WS based on both IHC and ISH was 97.7%. Only 14 tumours were discordant and one tumour with IHC score 1+ on both TMA and WS was HER2 amplified on TMA. In conclusion, GPA on TMA is suitable for large-scale retrospective evaluation of HER2 status.

Proteins are the essential components of the tissues that play a key role in the body. Its expression in the cell or tissue under a specified set of conditions and at a particular time regulates the different body conditions either as a normal body function or as a disease state. Protein is an important building block of muscles, skin, cartilage, bones and blood. Bradford assay is a reliable advanced and cost-effective protein estimation test for determining the exact concentration of protein in different tissues of the animal. In this study, we have taken a rat suffering from protein deficiency disorder and total protein concentration in the heart, brain, liver, blood and kidney was determined. It was found that the total protein concentration in different tissues of rat i.e., heart, brain, liver, plasma and kidney was found to be 8.39 ± 0.75, 10.46 ± 0.76, 6.74 ± 0.39, 8.12 ± 0.32 mg/g of tissue and 61.27 ± 0.95 mg/mL of plasma respectively (mean ± SEM). As compared to earlier published reports the total protein concentration in different tissues like hear, brain, liver and kidney found much lower to standard value as reported by Beyer, the reason behind obtaining this kind of results may be due to the presence of insufficient amount of the protein content in different tissue of animal as suffering from protein degeneration disorder. The rat was unable to digest and store the protein or catabolism was much faster than anabolism. Keywords: Anabolism, Bradford assay, Catabolism, Protein estimation.

Background: Amyloid fibrils are insoluble arranged aggregates of proteins that are fibrillar in structure and related to many diseases (at least 20 types of illnesses) and also create many pathologic conditions. Therefore understanding the circumstance of fibril formation is very important. Objectives: This study aims to work on fibrillar structure formation of fibroin (as a model protein). Material and Methods: In this experimental study, fibroin was extracted from bombyx mori silk cocoon, and the concentration was obtained by Bradford method. The protein was incubated in a wide range of times (0 min to 7 days) in specific acidity and thermal conditions (pH=1.6, T=70 °C). The assays of UV-vis spectroscopy with congo red, field emission scanning electron microscopy, transmission electron microscopy, atomic force microscopy and circular dichroism spectroscopy were employed to monitor the fibrillation process. Results: Fibroin assemblies were formed upon the process of aggregation and fibril formation with a variety of morphology ranging from nanoparticles to elongated fibrils. Conclusion: The results showed progressive pathway of fibril formation.

Rice (Oryza sativa L.) cultivated in Cameroon is appreciated by consumers for its nutritive quality and good taste. Diversity of 68 local rice cultivars was investigated via grain morphology and protein content characterization. The size and shape of grains were determined and used with yield parameters to classify the cultivars and perform Principal Component Analysis (PCA). Total protein content and glutelin content of eight selected cultivars (CMRGNd, CMRGDn, CMRGTi, CMRTBa, CMRDWb, CMRDTc3, CMRDTx5 and CMRDTx6) were evaluated by Bradford assay and correlation analysis of all the parameters studied was performed. Long size grains (42) were predominant over extra-long (16), medium (9) and short (1) grains. Slender shaped grains (36) were distinguished as well as medium (28) and bold (4) grains. The 68 cultivars were grouped into four clusters independent of their origins. PCA revealed three principal components accounting for 74.4% of total variation. Highest total protein content was observed in CMRGNd (14.3%) and highest glutelin content in CMRGDn (10.1 mgEqvBSA/g DW). Pearson correlation of the different variables revealed no significant correlation between total protein and glutelin contents with the agro-morphological parameters evaluated in this study. This suggests that none of these parameters could be descriptor for protein content. Positive correlation between grain length and yield (r = 0.7) suggests grain length as yield descriptor. Key words: Rice, diversity, grain morphology, protein, glutelin.

To evaluate the performance of the Ankylosing Spondylitis Disease Activity Score based on a validated quick quantitative C-reactive protein assay (ASDAS-qCRP) as compared to ASDAS based on a routine lab CRP assay (ASDAS-CRP) and ASDAS based on erythrocyte sedimentation rate (ASDAS-ESR).Disease activity assessment was performed in 50 patients with axial spondyloarthritis (axSpA). Routine lab CRP was measured in the central lab while the quantitative quick-CRP assay and ESR measurements were performed locally. ASDAS-CRP, ASDAS-qCRP and ASDAS-ESR were subsequently calculated.The mean (±SD) serum level of the routine lab CRP (6.2±8.3mg/l) was lower than of the quick-CRP (7.4±8.4mg/l) (P0.05). Whereat, there was no significant difference in the mean values of ASDAS-CRP and ASDAS-qCRP in axSpA patients (2.70±0.94 and 2.74±0.96, respectively, P=0.069), while the ASDAS-ESR (2.85±1.0) was significantly higher than ASDAS-CRP (P=0.036) and numerically higher than ASDAS-qCRP (P=0.125). In 47 of the 50 cases of axSpA (94%), patients were assigned to the same disease activity category according to ASDAS-CRP and ASDAS-qCRP.ASDAS-qCRP performed similarly well compared to ASDAS-CRP with the absolute agreement on the disease activity category according to the ASDAS of 94%. ASDAS-qCRP is, therefore, feasible for an immediate decision-making in clinical practice and trials aimed at treating to target.

Western blotting is a widely used protein assay platform, but the technique requires long analysis times and multiple manual steps. Microfluidic systems are currently being explored for increased automation and reduction of analysis times, sample volumes, and reagent consumption for western blots. Previous work has demonstrated that proteins separated by microchip electrophoresis can be captured on membranes by dragging the microchip outlet across the membrane. This process reduces the separation and transfer time of a western blot to a few minutes. To further improve the speed and miniaturization of a complete western blot, a microscale immunoassay with direct deposition of immunoassay reagents has been developed. Flow deposition of antibodies is used to overcome diffusion limited binding kinetics so that the entire immunoassay can be completed in 1 h with detection sensitivity comparable to incubation steps requiring 20 h. The use of low microliter/min flow rates with antibody reagents applied directly and locally to the membrane where the target proteins have been captured, reduced antibody consumption ~30-fold. The complete western blot was applied to the detection of GAPDH and β-Tubulin from A431 cell lysate.

Background/Aims: Accurate diagnosis and the effects of allergen-specific immu notherapy for pollinosis are greatly dependent on the potency and stability of the extract. This study aimed to examine factors, such as temperature and storage buffer composition, that affect the stability of allergen extracts from pollens of allergenic importance in Korea. Methods: We prepared four pollen allergen extracts from ragweed, mugwort, Japanese hop, and sawtooth oak, which are the most important causes of season al rhinitis in Korea. Changes of protein and major allergen concentration were measured over 1 year by Bradford assay, two-site enzyme-linked immunosorbent assay, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis after recon stitution of the lyophilized allergen extract in various buffers and stored at room temperature (RT, 18°C to 26°C) or refrigerated (4°C). Results: More than 90% of the original protein concentration in all four extracts examined was detected over 1 year when 50% glycerol was added and refrigerated, whereas 57.9% to 94.5% remained in the extracts at RT. The addition of 50% glyc erol to the storage buffer was found to prevent protein degradation at RT. Amb a 1, a major allergen of ragweed, was almost completely degraded in 9 weeks at RT when reconstituted in a buffer without 50% glycerol. However, 55.6% to 92.8% of Amb a 1 content was detected after 1 year of incubation at 4°C in all buffer condi tions except 0.3% phenol. Conclusions: Addition of 50% glycerol as well as refrigeration was found to be im portant in increasing the shelf-life of allergen extracts from pollens of allergenic importance.

We demonstrate the use of an evaporating, sessile droplet on a nonwetting substrate as a miniature micromixing device to conduct sample–dye reactions for absorbance-based colorimetry. The nonwetting substrate supports buoyancy-induced mixing inside the droplet for rapid completion of the measurement. The Bradford assay is used as a proof of concept, where a protein-containing sample is reacted with a reagent dye to measure the protein concentration. Viability of absorbance measurement through the droplet is first established using droplets in which the reactants are mixed prior to their deposition onto the substrate. In a second set of experiments involving in situ mixing, the reagent is directly added to a sessile droplet of the protein-containing sample, allowing the reactants to mix while the absorbance is being measured. Interplay between buoyancy-induced mixing, protein–reagent reaction, and protein adsorption onto the substrate leads to a complex temporal absorbance measurement signal. Videos corresponding to the signal data show that each of these mechanisms dominates during different phases of droplet evolution, causing a signal pattern containing peaks and valleys having a strong monotonic trend with the protein concentration. Overall, the second absorbance peak at which the reaction nears completion is the most sensitive to sample concentration. Heating of the substrate is demonstrated to dramatically speed up the mixing process. These protein concentration measurements, obtained with a simpler system and low reactant volumes, demonstrate that this droplet micromixing concept is a viable alternative to microtiter plates for colorimetric applications.

A microfluidic open circuit potential label-free protein assay was developed for the reagentless quantification of C-reactive protein (CRP), a model protein target, and further utilized to assess target-receptor binding kinetics. Generated sensors have very high baseline stabilities (

Within the last years pollen grains have gained increasing attention due to their cloud-forming potential. Especially the discovery that ice nucleating macromolecules (INMs) or subpollen particles (SPPs) obtained from pollen grains are able to initiate freezing has stirred up interest in pollen. INMs or SPPs are much smaller and potentially more numerous than pollen grains and could significantly affect cloud formation in the atmosphere.However, INMs and SPPs are not clearly distinguished. This has motivated the present study, which focuses on birch pollen and investigates the relationship between pollen grains, INMs and SPPs. According to the usage of the term SPPs in the medical fields, we define SPPs as the starch granules contained in pollen grains. We show that these insoluble SPPs are only obtained when fresh pollen grains are used to generate aqueous extracts from pollen. Due to the limited seasonal availability of fresh pollen grains almost all studies have been conducted with commercial pollen grains. To enable the investigation of the SPPs we develop an alternative extraction method to generate large quantities of SPPs from commercial pollen grains. We show that INM are not bonded to SPPs (i.e. can be washed off with water). Further, we find that purified SPP are not ice nucleation active: after several times of washing SPPs with ultrapure water the ice nucleation activity completely disappears. To our knowledge, this is the first study to investigate the ice nucleation activity of isolated SPPs.To study the chemical nature of the INMs, we use fluorescence spectroscopy. Fluorescence excitation-emission maps indicate a strong signal in the protein range (maximum around λex = 280 nm and λem = 330 nm) with all ice nucleation active samples. In contrast, with purified SPP the protein signal is lost. We also quantify the protein concentration with the Bradford assay. The protein concentration ranges from 77.4 μg mL-1 (Highly concentrated INM) to below 2.5 μg mL-1 (purified SPP). Moreover, we investigate the connection between proteins and ice nucleation activity by treating the ice nucleation active samples with subtilisin A and urea to unfold and digest the proteins. After this treatment the ice nucleation activity clearly diminished. The results indicate a linkage between ice nucleation activity and protein concentration. The missing piece of the puzzle could be a glycoprotein, which exhibits carboxylate functionalities, can bind water in tertiary structures and displays degeneration and unfolding of its secondary structure due to heat treatment or reaction with enzymes. Even though purified SPPs are not ice nucleation active they could act as carriers of INMs and distribute those in the atmosphere.Reference of the study: Burkart, J., Gratzl, J., Seifried, T. M., Bieber, P., and Grothe, H.: Isolation of subpollen particles (SPPs) of birch: SPPs are potential carriers of ice nucleating macromolecules, Biogeosciences, 18, 5751–5765, https://doi.org/10.5194/bg-18-5751-2021, 2021.

A TiO2/ZnO oxide system was proposed as a support for the immobilization of laccase from Trametes versicolor (LTV). The obtained TiO2/ZnO/LTV biocatalytic system was then applied in the decolorization/degradation of C.I. Reactive Black 5 and C.I. Acid Green 25 dyes. The efficiency of immobilization was evaluated based on catalytic properties (Bradford method, oxidation reaction of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) and physicochemical (spectroscopic, porous, electrokinetic) analysis. The immobilization process was carried out with high performance (99.4%). Immobilized laccase retained about 40% of its activity in the whole analyzed temperature range and after 10 reaction cycles. Immobilization efficiency was also indirectly confirmed by the presence of characteristic functional groups (–C–H and –C–O), nitrogen and carbon on the TiO2/ZnO/LTV biocatalytic surface, identified by spectroscopic analyses. The increase in the surface area to 126 m2/g, change of isoelectric point (2.0) and zeta potential ranges (from +12.0 to −20.0 mV) after the immobilization process were also observed. The results show that the designed biocatalytic system enables the removal of acid dyes (C.I. Reactive Black 5 and C.I. Acid Green 25) with high efficiency (99% and 70%, respectively). Mass spectroscopy analysis indicated possible degradation products formed by the cleavage of N=N and C–N bonds.

Protein assays show great importance in medical research and disease diagnoses. Liquid crystals (LCs), as a branch of sensitive materials, offer promising applicability in the field of biosensing. Herein, we developed an ultrasensitive biosensor for the detection of low-concentration protein molecules, employing LC-amplified optofluidic resonators. In this design, the orientation of LCs was disturbed by immobilized protein molecules through the reduction of the vertical anchoring force from the alignment layer. A biosensing platform based on the whispering-gallery mode (WGM) from the LC-amplified optofluidic resonator was developed and explored, in which the spectral wavelength shift was monitored as the sensing parameter. The microbubble structure provided a stable and reliable WGM resonator with a high Q factor for LCs. It is demonstrated that the wall thickness of the microbubble played a key role in enhancing the sensitivity of the LC-amplified WGM microcavity. It is also found that protein molecules coated on the internal surface of microbubble led to their interactions with laser beams and the orientation transition of LCs. Both effects amplified the target information and triggered a sensitive wavelength shift in WGM spectra. A detection limit of 1 fM for bovine serum albumin (BSA) was achieved to demonstrate the high-sensitivity of our sensing platform in protein assays. Compared to the detection using a conventional polarized optical microscope (POM), the sensitivity was improved by seven orders of magnitude. Furthermore, multiple types of proteins and specific biosensing were also investigated to verify the potential of LC-amplified optofluidic resonators in the biomolecular detection. Our studies indicate that LC-amplified optofluidic resonators offer a new solution for the ultrasensitive real-time biosensing and the characterization of biomolecular interactions. Supplementary Information The online version contains supplementary material available at 10.1186/s43074-021-00041-1.

The protein content of the cowpea has proved to be very variable, especially of the green grain. The aim of this study, therefore, was to obtain information on the influence of the environment and genetic variability on the total protein content (TPC) of green cowpea grain, as well as to evaluate the effects of production components on this variable. Sixteen cowpea genotypes were evaluated in two environments (Pentecoste and Acaraú) in the state of Ceará, in a simple factorial scheme (16 x 2) for eight production components and protein content. The grain was collected while still green (after filling was complete - stage R5) and protein quantification was carried out using the Bradford method. Path analysis was performed in order to identify possible relationships between the agronomic variables and the protein content. There was a different response from the genotypes for each environment and in the interaction between genotype and environment. In Pentecoste, the MNC00-303-09E strain was highlighted, with a TPC of 6.602%, while in Acaraú, the Paulistinha cultivar stood out with a TPC content of 15.154%. For the genotypes under evaluation, selection for total protein content should be made in Acaraú via direct selection of the green-pod-grain weight, 100-grain weight and green-pod weight, and in Pentecoste, via indirect selection of the green-pod weight and green-grain yield. RESUMO O teor de proteína em feijão-caupi tem se mostrado muito variável, especialmente para grãos verdes. Portanto, o objetivo deste trabalho foi obter informações sobre a influência do ambiente e a variabilidade genética para o teor de proteínas totais (TPT) em grãos verdes de feijão-caupi, bem como avaliar os efeitos de componentes de produção sobre essa variável. Foram avaliados dezesseis genótipos de feijão-caupi em dois ambientes (Pentecoste e Acaraú) no estado do Ceará, em esquema fatorial simples (16 x 2) para oito componentes de produção e teor de proteínas. Os grãos foram coletados verdes (após o completo enchimento - estádio R5) e a quantificação proteica foi realizada pelo método de Bradford. Realizou-se a análise de trilha a fim de identificar possíveis relações entre as variáveis agronômicas e o teor de proteínas. Verificou-se resposta diferenciada dos genótipos nos ambientes de cultivo e interação genótipos por ambientes. Para o ambiente Pentecoste, o destaque foi a linhagem MNC00-303-09E com 6,602% de TPT e em Acaraú para a cultivar Paulistinha, com 15,154%. A seleção para teor de proteína total dos genótipos avaliados deve ser feita em Acaraú por seleção direta dos componentes massa de grãos de vagem verde, massa de cem grãos e massa de vagem verde, e em Pentecoste por seleção indireta da massa de vagem verde e componente produtividade de grãos verdes.