Quantitative Determination Of Proteins Research Articles

Маркери активного запалення у хворих на хронічний бронхіт у поєднанні з інсулінорезистентністю

By us for diagnostics of activity of inflammatory process for patients by a chronic bronchitis (CB) in combination with insulintolerance the necessity of leadthrough of study of gemogramme and concentration of C-reactive protein was set for the whey of blood. Information of integral indexes of activity of inflammation (IAI) for patients with CB at presence of insulintolerance in the pe riod of intensifying, and also and period of remission at majority inspected remained higher than norm in relation to healthy people. Quantitative determination of C-reactive protein in the whey of blood for the patients of CB with insulintolerance along with determination of level of leucocytes and indexes of leukogramme is the laboratory marker of activity of inflammation in the period of intensifying in the bronkholegochnoy system.

Quantitative determination of matrix Gla protein (MGP) and BMP-2 during the osteogenic differentiation of human periodontal ligament cells

Background and objectiveMatrix Gla protein (MGP) has been recognized as a potent calcification inhibitor and a regulator for bone morphogenetic protein-2 (BMP-2). The periodontal ligament (PDL) is a non-mineralized connective tissue located between two mineralized tissues, the cementum and the alveolar bone. However, the mechanism by which PDL prevents mineralization has yet to be defined. This study aims to examine the expression pattern of MGP and BMP-2 during human periodontal ligament cells (hPDLCs) osteogenic differentiation in vitro, preliminarily exploring their roles in this process. Materials and methodshPDLCs were obtained and cultured in mineralizing medium. The expression of MGP and BMP-2 was confirmed by RT-PCR and immunofluorescence staining. In the process of osteogenic induction, alkaline phosphatase (ALP) activity, extracelluar calcium deposition, and mineralized nodules were measured. Quantitative real-time RT-PCR was performed to evaluate mRNA expression of MGP, BMP-2 and osteogenic marker genes, including ALP, bone sialoprotein (BSP), type I collagen (COLI), osteocalcin (OCN), and runt-related transcription factor 2 (Runx2). The protein expression of MGP and BMP-2 was analyzed by western blotting. ResultsCo-localization of MGP and BMP-2 was visualized in hPDLCs. After osteogenic induction, ALP activity, calcium deposition, mineralized nodules, and osteogenic marker genes were significantly up-regulated. mRNA expression of MGP and BMP-2 generally decreased, although MGP mRNA increased on day 14 and 21 compared with the control, and protein expression of MGP and BMP-2 was down-regulated. ConclusionOur results indicate that MGP might regulate hPDLCs osteogenic differentiation which might keep a potential relationship with BMP-2 in this process.

Quantitative determination of fat and total protein in milk based on visible light scatter

A new optical spectroscopic method for milk fat and total protein analysis has been developed. In contrast to the conventional approach that generally relies on the components’ absorption, the suggested method is based on the phenomenon of light scatter by fat and protein particles. This fundamental distinction enables shifting the measurement to the cost-effective visible and adjacent near infrared region (below 1000 nm), where the scatter strongly dominates.

ELISA Kit for Mustard Protein Determination: Interlaboratory Study

An interlaboratory study in 12 laboratories was performed to prove the validation of the ELISA method developed for the quantitative determination of mustard protein in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. This kit did not produce any false-positive results or cross-reactivity with in-house validation for a broad range of food matrixes with no detectable mustard protein. All participants obtained the Mustard ELISA kit with standard operational procedures, a list of samples, samples, and a protocol for recording test results. The study included 15 food samples and two spiked samples. Seven food matrix samples of zero mustard content and four samples with mustard declared as an ingredient showed mustard protein content lower than that of the first standard (0.42 mg/kg). Four samples with mustard declared as an ingredient revealed mustard protein content above 12.5 mg/kg (the highest standard). The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as an LOQ (1.8 mg mustard proteins/kg) and LOD (0.5 mg mustard proteins/kg), for the kit were calculated.

Quantitative protein determination for CYP induction via LC‐MS/MS

The Cytochrome P450 (CYP) proteins are a family of membrane bound proteins that function as a major metabolizing enzyme in the human body. Quantification of CYP induction is critical in determining the disposition, safety and efficacy of drugs in humans. Described is a gel-free, high-throughput LC-MS approach to quantitate the CYP isoforms 1A2, 2B6, 3A4 and 3A5 by measuring isoform specific peptides released by enzymatic digestion of the hepatocyte incubations. The method uses synthetic stable isotope-labeled peptides as internal standards and allows both relative and absolute quantification to be performed from hepatic microsomal preparations. CYP protein determined by this LC-MS method correlated well with the mRNA and activity for induced levels of CYP1A2, CYP2B6 and CYP3A4. Interestingly, a small fold change was observed for the induction of 3A5 with phenobarbital. The results were reproducible with an average CV less then 10% for repeat analysis of the sample. This LC-MS method offers a robust assay for CYP protein quantitation for use in CYP induction assays.

ELISA Kit for Determination of Egg White Proteins: Interlaboratory Study

An interlaboratory study was conducted in 11 laboratories to validate an ELISA method developed for the quantitative determination of egg white proteins (EWPs) in foods. The ELISA kit used for this study is based on sheep polyclonal antibody. It does not produce any false-positive results or cross-reactivity in a broad food matrix range with zero EWP content. All participants obtained the Egg ELISA Kit-native with standard operational procedure and the list of samples, as well as the samples and a protocol for recording test results. The study included 10 food samples. Four samples of food matrix with zero EWP content showed EWP content lower than the first standard (EWP content 0.5 mg/kg). One sample of food matrix with zero EWP content revealed EWP content higher than standard 3 (1.5 mg EWP/kg). Five food samples containing EWP as an ingredient tested positive and one negative. The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as LOQ (1.4 mg EWP/kg) and LOD (0.43 mg EWP/kg), were calculated for the kit.

Changes in myocardial connexin 43 during ventricular fibrillation

To observe changes in connexin 43 (Cx43) after ventricular fibrillation (VF) and the effects of rotigaptide (ZP123) on Cx43. Thirty domestic pigs were randomly assigned to three groups (10 in each group): sham group, model group and ZP123 group. VF was induced by an 80 V AC transthoracic shock for 5 seconds with the use of subcutaneous needles. Before the induction of VF, animals in ZP123 group were administered with ZP123 (1 μg/kg bolus+10 μg×kg(-1)×h(-1) dissolved in 50 ml normal saline and pumped for 15 minutes ). Those in model group received 50 ml normal saline pumped for 15 minutes. For pigs in sham group VF was not induced and no fluid was given. After 8 minutes of VF, animals were euthanized and myocardial tissues were harvested along the long axis of each left ventricular free wall. Immunofluorescence combined with laser scanning confocal microscope was used to detect the distribution of Cx43. Western blotting was used for quantitative determination of Cx43 protein expression. Immunofluorescence signals for Cx43 in sham group were strong and regularly distributed. In model group, Cx43 signals were weak and distributed in heterogeneity, while in ZP123 group, Cx43 signals were enhanced and their distribution were much more orderly. Compared with sham group, the percentage area and the optical densities (A value) of Cx43 fluorescence signals and Cx43 protein expression were significantly decreased in model group [the percentage area: (0.64±0.36)% vs.(1.27±0.19)%, A value: 15 201± 2 613 vs. 30 634±4 975, Cx43 protein expression: 0.72±0.08 vs. 0.97±0.07, all P<0.05]. The level of Cx43 expression in ZP 123 group [the percentage area (0.96±0.16)%, A value 22 100±4 404, Cx43 protein expression 0.82±0.04] was much higher than model group (all P<0.05). During VF, down-regulation of myocardial Cx43 expression occurred, which could be attenuated by administration of ZP123.

Amino-acid and lipid compositions of Gelrem preparation

Studies of the chemical composition of flowers and the extract of the tansy Tanacetum pseudachilleae C. Winkl., herbs and the extract of absinthe Artemisia absinthium L., and buds of the tree Caryophyllus aromaticus L. showed that the essential oils were the principal biologically active compounds responsible for the unique activity of the preparation [1, 2]. The amino-acid and fatty-acid compositions of the preparation Gelrem were studied in order to characterize fully the aforementioned composition. Quantitative determination of total proteins of Gelrem was performed using the Kjeldahl method and the Kaar–Kelly spectrophotometric method [3]. The analytical results showed that the total protein content of the preparation was 12.87%. Table 1 describes the quality of proteins as characterized by the amino-acid composition. The amino-acid composition of the preparation was analyzed after hydrolysis on a T 339 amino-acid analyzer [4]. It was found that it had high contents of glutamine, leucine, and arginine. Moreover, the dominant essential amino acids were leucine, tyrosine, phenylalanine, and lysine. Next, we determined the content of total lipids. Extraction by CHCl 3 :MeOH (5, 7:3) isolated the total lipids. The combined extracts were washed with aqueous CaCl 2 (0.05%) to remove ballast components. The solvent was distilled off. The lipid content was determined gravimetrically. The yield was 10.5%. The fatty-acid composition of the extract was found using the usual method [5]. Methyl esters of fatty acids were analyzed by GLC on a Chrom 5 chromatograph with a flame-ionization detector, a column (2.5 m 4 mm) packed with DEHS sorbent (10%) on Inerton N-AW, column temperature 192°C, vaporizer temperature 250°C, and N 2 carrier gas at 30 mL/min. Fatty acids were identified by comparing their retention times with those of standards. Table 2 presents the results of the GLC analysis.

The use of FTIR spectroscopy to assess quantitative changes in the biochemical composition of microalgae

A mid-infrared spectroscopic method was developed for the simultaneous and quantitative determination of total protein, carbohydrate and lipid contents of microalgal cells. Based on a chemometric approach, measured FTIR (Fourier transform infrared) spectra from algal cells were reconstructed by a partial least square algorithm, using the spectra of the reference substances to determine their relative contribution to the overall cell spectrum. From this specific absorption, absolute macromolecular cell composition [pg cell(-1)] can be calculated using calibration curves, which have been validated by independent biochemical methods. The future potential of this method for photosynthesis research is shown by its application to follow time-resolved changes in the cellular composition of microalgae during an illumination period of several hours. We show how the macromolecular composition can be investigated by FTIR spectroscopy methods. This can substantially increase the efficiency of screening processes like bioreactor monitoring and may be beneficial in metabolic engineering of algal cells.

Validation of end-point and real-time PCR methods for the rapid detection of soy allergen in processed products

This work describes the development and validation of two PCR methods, end-point and real-time PCR, for the detection of soy protein in a wide rage of foodstuffs. These techniques are reliable and sensitive, allowing detection of trace amounts of soybean in processed products. TaqMan real-time PCR was the simpler and more rapid process, with a higher potential for automation and, therefore, currently the most suitable screening method. To verify correct operation of the proposed methodology, ELISA was used for quantitative determination of soy protein. In addition, 35 meat, fish and bakery processed products, which could potentially contain soy but was not declared on the label, were tested for the presence of soy DNA using the proposed methods. The methodologies will be valuable in issues regarding the presence of soy protein in processed products, especially in verifying labelling and security regulations to protect consumer's rights.

Quantification of tear proteins by SDS-PAGE with an internal standard protein: A new method with special reference to small volume tears

Quantitative determination of tear proteins is critical to our understanding of those ocular diseases with tear protein changes, but remains technically complex due to the small sample volumes available from patients. The aim of this study was to efficiently quantify the tear proteins by SDS-PAGE with an internal standard protein in small tear volumes. Schirmer test paper and capillary tubes were used to collect tear samples. Soybean trypsin inhibitor (SBTI) was used as an external standard or an internal standard to analyze tear samples in 15% SDS-PAGE gel. The total tear protein and its major components were quantified by band densitometry. Total tear protein concentrations were also measured by Bradford assay. Using this internal standard method, we compared differences between tear samples collected by the Schirmer test paper and capillary tube, and then examined the differences detected between tear samples obtained from young and elderly people. Using SBTI as an internal standard in SDS-PAGE, the total tear protein concentrations were determined to be 12.03 +/- 0.45 mg/ml, showing no difference from the result determined by the Bradford assay (P > 0.05). The quantities of the eight major tear protein bands were 2.26 +/- 0.07(18.8%), 0.10 +/- 0.01(0.8%), 0.63 +/- 0.13(5.3%), 0.52 +/- 0.07(4.3%), 1.14 +/- 0.18(9.5%), 1.33 +/- 0.21(11.1%), 2.76 +/- 0.16(23.0%), and 2.95 +/- 0.13 mg/ml (24.5%), similar to the result obtained by using SBTI as an external standard (P > 0.05). Comparing the methods of collection, we identified that the Schirmer test paper sampling induced increased concentrations of band 2 (mainly HSA) in tears, and decreased five other major tear protein bands. Comparing total protein concentrations among different age groups, we noted a higher total protein concentration in young people (P < 0.05). This high protein level was contributed equally by all major tear protein components. Using SBTI as an internal standard, we can simultaneously quantify total tear protein and the major tear protein components by SDS-PAGE densitometry using small volume tears. This method appears promising for use as a diagnostic tool for identifying the occurrence of ocular diseases with tear protein changes.

Single Domain Antibodies Are Specially Suited for Quantitative Determination of Gliadins under Denaturing Conditions

Food intended for celiac patients' consumption must be analyzed for the presence of toxic prolamins using high detectability tests. Though 60% ethanol is the most commonly used solvent for prolamins extraction, 2-mercaptoethanol (2-ME) and guanidinium chloride (GuHCl) can be added to increase protein recovery. However, ethanol and denaturing agents interfere with antigen recognition when conventional antibodies are used. In the present work, a new method for gliadins quantification is shown. The method is based on the selection of llama single domain antibody fragments able to operate under denaturing conditions. Six out of 28 VHH-phages obtained retained their binding capacity in 15% ethanol. Selected clones presented a long CDR3 region containing two additional cysteines that could be responsible for the higher stability. One of the clones (named VHH26) was fully operative in the presence of 15% ethanol, 0.5% 2-ME, and 0.5 M GuHCl. Capture ELISA using VHH26 was able to detect gliadins in samples shown as negatives by conventional ELISA. Therefore, this new strategy appears as an excellent platform for quantitative determination of proteins or any other immunogenic compound, in the presence of denaturing agents, when specific recognition units with high stability are required.

Interaction of Tumor Suppressor p53 with DNA and Proteins

p53, a tumor suppressor and a transcription factor, binds DNA in a sequence-specific manner. In more than half of human cancers, p53 has been found to be mutated with the loss of DNA-binding ability. In this review, we focus on the sensitive detection of interaction of tumor suppressor p53 with double-stranded DNA bearing the consensus sequence and proteins, such as monoclonal antibodies recognizing the p53 protein and metalloprotein. Relying on the specific binding of p53 to DNA and antibodies, quantitative determination of wild-type and mutant p53 proteins in normal and cancer cell lysates has been achieved.

Temperature and Denaturing Substances Influence on Lab-on-a-Chip Electrophoresis

Temperature and Denaturing Substances Influence on Lab-on-a-Chip Electrophoresis Qualitative and quantitative determination of proteins in different biological fluids is of great significance in medicine, due to their importance in diagnosis and treatment of some diseases. Nowadays, different methods for protein analysis are available. Lab-on-a-chip electrophoresis is a relatively new technique, based on microfluidics, which allows samples of biological fluids to be analyzed within a microchip. This paper describes the optimization of performance of the chip-based protein analyses in serum samples from patients with different neurological disorders. Using microchip technology, serum proteins with the molecular mass from 4.5 to 240 Kb were separated and sized. The fluorescence detection method in the analysis was used to follow the influence of the temperature and the type and concentration of denaturing substances on the electrophoresis protein profiles. It was noted that, depending on incubation temperature and denaturing substances, different electrophoresis patterns can be obtained from the proteins of one specimen. Significant change of the fluorescence intensity was observed when different incubation temperatures were used, probably due to fluorescence quenching. In some cases, the band intensity was changed several times. Lab-on-a-chip electrophoresis is a very efficient method for the separation and determination of different serum proteins in a very short time. However, to obtain comparable results for the analysis, the denaturing agent concentration and temperature must be observed and maintained carefully.

Platinum Nanoparticle–Based Assay for Proteins by Resonance Light Scattering

ABSTRACT A simple, sensitive, platinum nanoparticle–based assay for trace amounts of protein by resonance light scattering (RLS) was studied. The RLS intensities for bovine serum albumin (BSA) and human serum albumin (HSA) were remarkably enhanced due to the interaction with Pt nanoparticles at pH 3.5 or 4.0, respectively, and the maximum RLS peak appeared at 390 nm. The RLS intensity increased proportionally with Pt concentration below 3.0 × 10−5 mol · L−1 but declined gradually above 4.0 × 10−5 mol · L−1. BSA within the range 0.1–3.0 µg · mL−1 and HSA of 0.1–2.7 µg · mL−1 can be detected with this method, and the detection limits were 4.2 and 10.7 ng · mL−1, respectively. The method was satisfactorily applied to the quantitative determination of total proteins in human serum samples with a maximum relative standard deviation (RSD) lower than 2.8% and recoveries in the range 100.4–102%.

Artificial neural network for quantitative determination of total protein in yogurt by infrared spectrometry

A method has been introduced for quantitative determination of protein content in yogurt samples based on the characteristic absorbance of protein in 1800–1500 cm − 1 spectral region by mid-FTIR spectroscopy and chemometrics. Successive Projection Algorithm (SPA) wavelength selection procedure, coupled with feed forward Back-Propagation Artificial Neural Network (BP-ANN) model was the benefited chemometric technique. Relative Error of Prediction (REP) in BP-ANN and SPA-BP-ANN methods for training set was 7.25 and 3.70 respectively. Considering the complexity of the sample, the ANN model was found to be reliable, while the proposed method is rapid and simple, without any sample preparation step.

Ultra Performance Liquid Chromatography Isotope Dilution Tandem Mass Spectrometry for the Absolute Quantification of Proteins and Peptides

A selective, rapid, and sensitive 12.7-min ultra performance liquid chromatography-isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) method was developed and compared to conventional high-performance liquid chromatography-isotope dilution tandem mass spectrometry (HPLC-ID/MS/MS) for the absolute quantitative determination of multiple proteins from complex matrixes. The UPLC analysis was carried out on an Acquity UPLC ethylene-bridged hybrid (BEH) C18 reversed-phase column (50 x 2.1 mm i.d., 1.7-microm particle size) with gradient elution at a flow rate of 300 microL/min. For the HPLC separation, a similar gradient profile on a reversed-phase C18 column with dimensions of 150 x 1.0 mm at a flow rate of 30 microL/min was utilized. The aqueous and organic mobile phases were 0.1% formic acid in water and acetonitrile, respectively. Detection was performed on a triple-quadrupole mass spectrometer operated in the multiple reaction monitoring mode. Linear calibration curves were obtained in the concentration range of 10-90 fmol/microL. Relative standard deviation values equal to or less than 6.5% were obtained by the UPLC-ID/MS/MS method, thus demonstrating performance equivalent to conventional HPLC-ID/MS/MS for isotope dilution quantification of peptides and proteins. UPLC provides additional dimensions of rapid analysis time and high-sample throughput, which expands laboratory emergency response capabilities over conventional HPLC.

A Metal-coded Affinity Tag Approach to Quantitative Proteomics

The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures.

Rapid quantitative determination of C-reactive protein at chair side in dental emergency patients

The objective of this study was to quantitatively determine, at chair side, the serum C-reactive protein (CRP) levels in dental emergency patients. Quantitative CRP test was performed at chair side in 40 patients with acute alveolar abscess (AAA), acute periodontal abscess (APA), and alveolar osteitis (AO) at the time of dental emergency treatment and 1 week after. CRP levels were compared between groups and before and after treatments using ANOVA and Fisher's Exact tests. Serum CRP levels were greater than 5 mg/L in 30 (75%) of the 40 patients. At 1-week follow-up, the decline in CRP levels was evident in the AAA group (P < .05), but not statistically significant in the APA and AO groups (P > .05). Serum CRP levels are often elevated in patients with odontogenic infections and postoperative complications. Rapid reduction in serum CRP levels was likely to occur following successful treatment of AAA, but less likely to occur in APA and AO.

Scopoletine as fluorescence probe for determination of protein

Scopoletine (SLT), 7-hydroxy-6-methoxylcoumarin, is known to possess biological activities such as abirritating and anti-tumor, it can quench intrinsic fluorescence of bovine serum albumin (BSA) and the fluorescence intensity of itself is enhanced. So, SLT is used as fluorescence probe for quantitative determination of protein. The experiments indicate that under optimum conditions, the enhanced intensity of fluorescence is in proportion to the concentration of proteins in a wide range, and their detection limits (S/N = 3) are 1.4 × 10 −8 g mL −1 for BSA and 1.1 × 10 −8 g mL −1 for HSA, respectively. Samples were satisfactorily determined. The interaction mechanism is also discussed.