This paper proposes a methodology for the classification and determination of total protein in milk powder using near infrared reflectance spectrometry (NIRS) and variable selection. Two brands of ...milk powder were acquired from three Brazilian cities (Natal-RN, Salvador-BA and Rio de Janeiro-RJ). The protein content of 38 samples was determined by the Kjeldahl method and NIRS analysis. Principal component regression (PCR) and partial least squares (PLS) multivariate calibrations were used to predict the total protein. Soft independent modeling of class analogy (SIMCA) was also used for full-spectrum classification, resulting in almost 100% classification accuracy, regardless of the significance level adopted for the
F-test. Using this strategy, it was feasible to classify powder milk rapidly and nondestructively without the need for various analytical determinations. Concerning the multivariate calibration models, the results show that PCR, PLS and MLR-SPA models are good for predicting total protein in powder milk; the respective root mean square errors of prediction (RMSEP) were 0.28 (PCR), 0.25 (PLS), 0.11
wt% (MLR-SPA) with an average sample protein content of 8.1
wt%. The results obtained in this investigation suggest that the proposed methodology is a promising alternative for the determination of total protein in milk powder.
•Paired LEDs are effective as turbimetric as well as nephelometric detectors.•The detectors are easily adapted for flow analysis.•Exton method with PEDDs under FIA conditions is useful for ...urinalysis.
Two miniature and compact optoelectronic devices fabricated by means of integration of light emitting diodes have been developed for turbidimetric and nephelometric measurements. These devices are operating according to paired-emitter-detector-diode (PEDD) principle. The detectors have been characterized using bovine serum albumin and Exton protein assay as a model analyte and a model analytical method, respectively. The developed detectors have been adapted for measurements under conditions of flow injection analysis (FIA). Under optimized conditions the turbidimetric flow system offers the range of linear response up to 400mgL−1 with the detection limit at 20mgL−1. The linear range and detection limit found for optimized nephelometric FIA system are 15–500mgL−1 and 8mgL−1, respectively. The PEDD-based FIA systems with the detector operating according to both modes of measurements have been successfully applied for urinalysis offering total protein determination at physiological and pathological levels with high throughput (over 60 injections per hour).
Ultrasound-based protein determination in maize seeds Drochioiu, Gabi; Ciobanu, Catalina Ionica; Bancila, Sabina ...
Ultrasonics sonochemistry,
March 2016, 2016-Mar, 2016-03-00, 20160301, Volume:
29
Journal Article
Peer reviewed
Open access
•Ultrasound-assisted extraction and determination of proteins in cereal seeds were investigated.•The amount of protein extracted is time and particle size dependent.•The biuret-determined protein ...values are in excellent agreement with those obtained by the Micro-Kjeldahl method.
The need for a simple and accurate method for protein estimation in alcoholic extracts led to the reexamination of the optimum conditions of a colorimetric assay based on the biuret reaction. Sonication time and the other experimental parameters were optimized after kinetics study on the extraction of either zein or total proteins. Zein extraction and purity were investigated by 1H and 13C NMR spectroscopy, SDS–PAGE electrophoresis, and UV–visible spectrophotometry (UV–vis). A zein assay was proposed, which involves the reaction of copper ions in copper phosphate powder with zein extracted in ethanolic solutions under strong alkaline environment. Furthermore, we extended this procedure to determine total proteins in maize samples simultaneously with their ultrasonic-assisted (US) extraction with an alkaline–alcoholic solution. Proteins in both types of extracts were well characterized by UV–vis spectroscopy. However, the 545nm absorbance of the violet-colored supernatants which is proportional to the protein content was found to be the key parameter of the improved biuret-based protein assay. Comparison of values obtained by this procedure and by Micro-Kjeldahl method was in excellent agreement. A scaled-down procedure agreed well with the standard procedure. Enhanced accuracy and repeatability was found in protein determination in maize using the modified biuret method. The optimization of reagent concentrations and incubation times were studied as well.
More than 30 proteins and peptides have been found to form amyloid fibrils in human diseases. Fibrils formed by transthyretin (TTR) are associated with ATTR amyloidosis, affecting many vital organs, ...including the heart and peripheral nervous system. Congo red staining is the gold standard method for detection of amyloid deposits in tissue. However, Congo red staining and amyloid typing methods such as immunofluorescence labelling are limited to relatively large deposits. Detection of small ATTR deposits present at an early stage of the disease could enable timely treatment and prevent severe tissue damage. In this study, we developed an enhanced ATTR amyloid detection method that uses functionalised protein nanofibrils. Using this method, we achieved sensitive detection of monomeric TTR in a microplate immunoassay and immunofluorescence labelling of ex vivo tissue from two patients containing ATTR aggregates. The system's utility was confirmed on sections from a patient with AA amyloidosis and liver sections from inflamed mouse. These results suggest that the detection system constitutes important new technology for highly sensitive detection of microscopic amounts of ATTR amyloid deposited in tissue.
•We establish a rapid method for determining protein content in dairy products.•We examine effects of different conditions on results.•Main merit of our approach is it can avoid interferences of ...other nitric-compounds.•Other features are that reproducibility is better and analysis speed is rapid.•It is applicable for determining the samples containing protein in many areas.
An accurate and rapid method and a system to determine protein content using asynchronous-injection alternating merging zone flow-injection spectrophotometry based on reaction between coomassie brilliant blue G250 (CBBG) and protein was established. Main merit of our approach is that it can avoid interferences of other nitric-compounds in samples, such as melamine and urea. Optimized conditions are as follows: Concentrations of CBBG, polyvinyl alcohol (PVA), NaCl and HCl are 150mg/l, 30mg/l, 0.1mol/l and 1.0% (v/v), respectively; volumes of the sample and reagent are 150μl and 30μl, respectively; length of a reaction coil is 200cm; total flow rate is 2.65ml/min. The linear range of the method is 0.5–15mg/l (BSA), its detection limit is 0.05mg/l, relative standard deviation is less than 1.87% (n=11), and analytical speed is 60 samples per hour.
Effective development of production of any therapeutic protein requires a relatively fast, reliable, and economical method of target protein monitoring in the outlet streams of cultivation and ...separation steps. The aim of this work was to develop a method for the determination of recombinant human erythropoietin (rhEPO) produced by human embryonic kidney (HEK) cells. An indirect ELISA method was designed that meets all requirements to fulfill this goal. It was found that an HEK cell-cultivation medium, containing plant hydrolyzate, is a suitable coating buffer, which solves the problem of competitive binding of byproducts. The method has a linear range of 0.078–4 μg/mL allowing accurate determination of rhEPO in relatively concentrated solutions (about 200 μg/mL) without excessive dilution. For the multicomponent rhEPO concentrate at the 400-fold dilution, the inter-assay coefficient of variation was found to be 3.3%. The cost analysis showed that the designed indirect ELISA assay is by one–two orders of magnitude cheaper than the existing commercial sandwich ELISA kits.
Abstract Celia's Encephalopathy (MIM # 615924 ) is a recently discovered fatal neurodegenerative syndrome associated with a new BSCL2 mutation (c.985C>T) that results in an aberrant isoform of seipin ...(Celia seipin). This mutation is lethal in both homozygosity and compounded heterozygosity with a lipodystrophic BSCL2 mutation, resulting in a progressive encephalopathy with fatal outcomes at ages 6–8. Strikingly, heterozygous carriers are asymptomatic, conflicting with the gain of toxic function attributed to this mutation. Here we report new key insights about the molecular pathogenic mechanism of this new syndrome. Intranuclear inclusions containing mutant seipin were found in brain tissue from a homozygous patient suggesting a pathogenic mechanism similar to other neurodegenerative diseases featuring brain accumulation of aggregated, misfolded proteins. Sucrose gradient distribution showed that mutant seipin forms much larger aggregates as compared with wild type (wt) seipin, indicating an impaired oligomerization. On the other hand, the interaction between wt and Celia seipin confirmed by coimmunoprecipitation (CoIP) assays, together with the identification of mixed oligomers in sucrose gradient fractionation experiments can explain the lack of symptoms in heterozygous carriers. We propose that the increased aggregation and subsequent impaired oligomerization of Celia seipin leads to cell death. In heterozygous carriers, wt seipin might prevent the damage caused by mutant seipin through its sequestration into harmless mixed oligomers.
The solid-phase protein assays using blotting membranes as hard support do not allow achieving the low background and sensitivity of protein staining in clear gels. The membrane opacity complicates ...imaging of results on standard lab documentation systems. We describe a low-cost transparent matrix that can be used as an alternative to polymeric membranes for solid-phase assays. Protein samples are spotted onto a dry film of composite agarose–polyacrylamide matrix covering standard glass microscopic slides. After rehydration in protein-fixing solution, matrix with protein samples can be detached from glass support and stained as conventional protein polyacrylamide gels.
A fully mechanized, computer-controlled, multicommutated flow analysis (MCFA) system dedicated for total protein determination in cerebrospinal fluid samples has been developed. For the protein ...determination the Exton method has been applied. Dedicated turbidimetric and nephelometric flow-through detectors operating according to paired-emitter detector diode principle have been fabricated by integration of two or three respective light emitting diodes. The developed MCFA system is characterized by robust, compact design and low consumption of the sample (72μL). The limits of detection for turbidimetric and nephelometric detection mode are 65mgL−1 and 9mgL−1, respectively. For turbidimetric measurements the range of linear response offered by the MCFA system is 72–900mgL−1, whereas in the case of nephelometric detection 18–500mgL−1 linear range is obtained. The throughput of the MCFA system is over 30 injection per hour. The analytical system was optimized with bovine serum albumin standards and successfully validated with real samples of human cerebrospinal fluid.
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•Multicommutated flow analysis system for total protein determination has been developed.•For turbidimetric and nephelometric measurements an integrated optoelectronic detector has been developed.•The MCFA system is useful for analysis of cerebrospinal fluid.
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