scholarly journals Widely used commercial ELISA does not detect preHP-2, but recognizes properdin as a potential second member of the zonulin family

2017 ◽  
Author(s):  
Lucas Scheffler ◽  
Alyce Crane ◽  
Henrike Heyne ◽  
Anke Tönjes ◽  
Dorit Schleinitz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Intestinal Permeability ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Metabolic Diseases ◽  
Polyclonal Antibodies ◽  
Complement C3 ◽  
Elisa Kit ◽  
Haptoglobin Genotype

AbstractBACKGROUNDThere is increasing evidence for the role of impaired intestinal permeability in obesity and associated metabolic diseases. Zonulin is an established serum marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to investigate the relationship between circulating zonulin and metabolic traits related to obesity.METHODSSerum zonulin was measured by using a widely used commercial ELISA kit in 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In addition, haptoglobin genotype was determined in DNA samples from all study subjects.RESULTSAs zonulin concentrations did not correlate to the haptoglobin genotypes, we investigated the specificity of the zonulin ELISA assay using antibody capture experiments, mass spectrometry and Western blot analysis. Using serum samples that gave the highest or lowest ELISA signals, we detected several proteins that are likely to be captured by the antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. We used increasing concentrations of recombinant pre-haptoglobin 2 and complement C3 as one of the representative captured proteins and the ELISA kit did not detect either. Western blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies rose against zonulin showed a similar protein recognition pattern but with different intensity of detection. The protein(s) measured using the ELISA kit was (were) significantly increased in patients with diabetes and obesity and correlated strongly with markers of the lipid and glucose metabolism. Combining mass spectrometry and Western blot analysis using the polyclonal antibodies used in the ELISA kit, we identified properdin as another member of the zonulin family.CONCLUSIONSOur study suggests that the zonulin ELISA does not recognize pre-haptoglobin 2, rather structural (and possibly functional) analogue proteins belonging to the mannose-associated serine protease family, with properdin being the most likely possible candidate.

scholarly journals Proteomic profiles of unilateral cryptorchidism in pigs at different ages using MALDI-TOF mass spectrometry and in-gel digestion coupled with mass spectrometry (GeLC-MS/MS) approaches

2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Nathamon Yimpring ◽  
Sittiruk Roytrakul ◽  
Janthima Jaresitthikunchai ◽  
Narumon Phaonakrop ◽  
Sucheewin Krobthong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Expression Profiles ◽  
Tumor Necrosis Factor Receptor ◽  
Principal Component ◽  
Peptide Mass ◽  
Different Ages

Abstract Background Cryptorchidism is a condition that occurs when one or both testes fail to descend into the scrotum. It is a common congenital disorder, causing economic loss in pig production. However, there have been only limited studies of differential protein expression profiles in undescended testes (UDTs) in the abdomen and descended testes (DTs) in cryptorchid pigs, especially at the peptidome and proteome levels. The present study aimed to analyze the peptidome of UDTs and DTs in unilateral cryptorchid pigs aged 1–2, 6, 15 and 20 weeks and in normal testes of healthy pigs aged 1–2 and 12 weeks, using peptide mass fingerprinting and three-dimensional principal component analysis (3D-PCA) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and to identify potential protein candidates, using in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). Western blot analysis was used to verify protein expression. Protein sequence was affirmed by liquid chromatography–tandem mass spectrometry. Results A PCA plot showed a discrete cluster for each sample group. Peptide mass fingerprints (PMFs) demonstrated unique peptide fragments in UDTs at different ages. A number of markedly expressed proteins from GeLC-MS/MS were identified, including the multifunctional tumor necrosis factor receptor superfamily member 18 (TNFRSF18), in DTs at 1–2 and 6 weeks and in UDTs at 15 and 20 weeks of age. Using western blot analysis, high expression of TNFRSF18 was observed in the UDTs at 15 weeks. Using the STITCH database, this protein was found to be related to apoptosis, corresponding to the previous report in the UDTs at the same age. Conclusions The present study revealed the specific PMFs and clusters for porcine cryptorchidism, and a novel protein, TNFRSF18, associated with the disease mechanism. These results could provide further insights into the pathogenesis of the disease.

scholarly journals Identification of cytoplasmic sialidase NEU2-associated proteins by LC-MS/MS

2018 ◽  
Vol 44 (4) ◽  
pp. 462-472
Author(s):  
Secil Akyildiz Demir ◽  
Volkan Seyrantepe
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Blot Analysis ◽  
Actin Filaments ◽  
Western Blot Analysis ◽  
Mammalian Cells ◽  
Protein S ◽  
Mass Spectrometry Analysis ◽  
Interacting Protein ◽  
Spectrometry Analysis

Abstract Background Cytoplasmic sialidase (NEU2) plays an active role in removing sialic acids from oligosaccharides, glycopeptides, and gangliosides in mammalian cells. NEU2 is involved in various cellular events, including cancer metabolism, neuronal and myoblast differentiation, proliferation, and hypertrophy. However, NEU2-interacting protein(s) within the cell have not been identified yet. Objective The aim of this study is to investigate NEU2 interacting proteins using two-step affinity purification (TAP) strategy combined with mass spectrometry analysis. Methods In this study, NEU2 gene was cloned into the pCTAP expression vector and transiently transfected to COS-7 cells by using PEI. The most efficient expression time of NEU2- tag protein was determined by real-time PCR and Western blot analysis. NEU2-interacting protein(s) were investigated by using TAP strategy combined with two different mass spectrometry experiment; LC-MS/MS and MALDI TOF/TOF. Results Here, mass spectrometry analysis showed four proteins; α-actin, β-actin, calmodulin and histone H1.2 proteins are associated with NEU2. The interactions between NEU2 and actin filaments were verified by Western blot analysis and immunofluorescence analysis. Conclusions Our study suggests that association of NEU2 with actin filaments and other protein(s) could be important for understanding the biological role of NEU2 in mammalian cells.

scholarly journals Selection and Characterization of Conditionally Active Promoters in Lactobacillus plantarum, Using Alanine Racemase as a Promoter Probe

2004 ◽  
Vol 70 (1) ◽  
pp. 310-317 ◽  
Author(s):  
Peter A. Bron ◽  
Sally M. Hoffer ◽  
Iris I. Van Swam ◽  
Willem M. De Vos ◽  
Michiel Kleerebezem
Keyword(s):  
Western Blot ◽  
Lactobacillus Plantarum ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Polyclonal Antibodies ◽  
Screening Method ◽  
Plasmid Vector ◽  
Alanine Racemase ◽  
Gene Encoding ◽  
Plasmid Library

ABSTRACT This paper describes the use of the alr gene, encoding alanine racemase, as a promoter-screening tool for the identification of conditional promoters in Lactobacillus plantarum. Random fragments of the L. plantarum WCFS1 genome were cloned upstream of the promoterless alr gene of Lactococcus lactis in a low-copy-number plasmid vector. The resulting plasmid library was introduced into an L. plantarum Δalr strain (MD007), and 40,000 clones were selected. The genome coverage of the library was estimated to be 98%, based on nucleotide insert sequence and restriction analyses of the inserts of randomly selected clones. The library was screened for clones that were capable of complementing the d-alanine auxotroph phenotype of MD007 in media containing up to 10, 100, or 300 μg of the competitive Alr inhibitor d-cycloserine per ml. Western blot analysis with polyclonal antibodies raised against lactococcal Alr revealed that the Alr production level required for growth increased in the presence of increasing concentrations of d-cycloserine, adding a quantitative factor to the primarily qualitative nature of the alr complementation screen. Screening of the alr complementation library for clones that could grow only in the presence of 0.8 M NaCl resulted in the identification of eight clones that upon Western blot analysis showed significantly higher Alr production under high-salt conditions than under low-salt conditions. These results established the effectiveness of the alanine racemase complementation screening method for the identification of promoters on their conditional or constitutive activity.

scholarly journals Identification of Immmunogenic Salivary Proteins of Anopheles vagus based on Mass Spectrometry Analysis

2017 ◽  
Vol 18 (2) ◽  
pp. 73
Author(s):  
Dwi Esti Febriyantiningsih ◽  
Kartika Senjarini ◽  
Rike Oktarianti
Keyword(s):  
Mass Spectrometry ◽  
Salivary Gland ◽  
Western Blot ◽  
Salivary Glands ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mass Spectrometry Analysis ◽  
Salivary Proteins ◽  
Immunogenic Proteins ◽  
Anopheles Vagus

Malaria has been prevalent for a long time in tropical developing regions causing great morbidity and mortality. Among the malaria vectors, Anopheles vagus has been known as secondary malaria vector in East Java. Salivary glands of mosquitoes perform various functions for survival of the vectors and also conducive for blood feeding, harbouring of malaria parasites, and eventual parasite transmission. The salivary gland proteomes of An. vagus have not been carried out yet. The aim of our study was to identify and characterize the immunogenic proteins of salivary glands proteins of An. vagus. A proteomic approach combining one-dimensional electrophoresis (1DE) followed by western blot analysis using human sera from healthy people living in an endemic area (Kendal); liquid chromatography mass spectrometry (LC-MS/MS) and bioinformatic analysis was adopted to provide the first direct insight into identification and characterization of salivary proteins of An. vagus. Identification of immunogenic proteins using western blot analysis has revealed three immunogenic bands which had molecular weights of 69, 75 and 232 kDa. Among those proteins analysed by LC-MS/MS, there were alpha,1-4 glucan phosphorylase, putative myosin class I heavy chain which have the highest number of total spectrum count peptide. Other proteins like vitellogenin and heat shock protein 82 (Hsp82) were also identified. The majority of proteins were scrutinized marked for their role in metabolism, cytoskeleton protein and stress response. Keywords: Anopheles vagus, salivary gland, immunogenic, proteomics

scholarly journals CRALBP is a Highly Prevalent Autoantigen for Human Autoimmune Uveitis

2007 ◽  
Vol 2007 ◽  
pp. 1-6 ◽  
Author(s):  
Cornelia A. Deeg ◽  
Albert J. Raith ◽  
Barbara Amann ◽  
John W. Crabb ◽  
Stephan R. Thurau ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Two Dimensional ◽  
Autoimmune Uveitis ◽  
Equine Recurrent Uveitis ◽  
And Control ◽  
Normal Controls ◽  
Subsequent Identification

Cellular retinaldehyde binding protein (CRALBP) is an autoantigen in spontaneous equine recurrent uveitis. In order to test whether CRALBP contributes to human autoimmune uveitis, the specificity of antibodies from human uveitis patient's sera was first evaluated in two-dimensional (2D) Western blot analysis. Subsequent identification of the immunoreactive proteins by mass spectrometry resulted in the identification of CRALBP as a putative autoantigen. Additionally, sera from human uveitis and control patients were by Western blot using purified human recombinant CRALBP. Anti-CRALBP autoantibodies occur more frequently (P<.01) in human uveitis patients than in normal controls. Thirty out of 56 tested uveitis patient's sera contained autoantibodies reactive against CRALBP, compared to only four out of 23 normal control subjects. The presence of CRALBP autoantibodies in 54% of tested uveitis patients supports CRALBP as a possible autoantigen in human autoimmune uveitis.

scholarly journals Overproduction and Purification of Soluble Recombinant Human Granulocyte Colony Stimulating Factor in Escherichia coli Using Thioredoxin as Fusion

2017 ◽  
Vol 21 (1) ◽  
pp. 1
Author(s):  
Dian Fitria Agustiyanti ◽  
Debbie Sofie Retnoningrum ◽  
Heni Rachmawati ◽  
Asrul Muhamad Fuad
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Soluble Form ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor

Recombinant human Granulocyte Colony Stimulating Factor (G-CSF) has been produced in a soluble form in Escherichia coli BL21 (DE3) as a fusion protein. The open reading frame of G-CSF was synthetically constructed in previous work and was codon optimized for best expression in E. coli. In this research, the gene was fused to thioredoxin (Trx) at the N-terminal in pET32 vector. The purpose of this research was to optimize the overproduction and purification processes to obtain high yield recombinant protein in soluble form, and to characterize the Trx-G-CSF fusion protein. Overproduction was performed using IPTG induction method for 3 and 6 hours. The protein was purified by Ni-NTA affinity chromatography and separated using gradient concentration of imidazole. The purified protein was then characterized by SDS-PAGE and Western Blot analysis. Further, enterokinase was used to separate G-CSF from the fusion protein. The purified form of G-CSF was subsequently characterized using Western Blot and mass spectrometry using MALDI-TOF. The results showed that the fusion protein was successfully produced in soluble part as much as 48.25% were obtained after 3 hours of induction. The yield of  fusion protein was 67.37%  from total protein (229.65  mg protein/L culture). The Western Blot analysis showed the G-CSF band at around 18.6 kDa. Mass spectrometry with MALDI-TOF/ TOF revealed that 25.86% of amino acid residue was recognized as part of human G-CSF sequence. 

The porcine glucocorticoid receptor: sequencing a 2.1 kb cDNA fragment and raising specific polyclonal antibodies for western blotting, immunoprecipitation and immunohistochemistry

2001 ◽  
Vol 73 (2) ◽  
pp. 267-278 ◽  
Author(s):  
M. Gutscher ◽  
S. Eder ◽  
M. Müller ◽  
R. Claus
Keyword(s):  
Glucocorticoid Receptor ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Polyclonal Antibodies ◽  
Cdna Fragment ◽  
E Coli ◽  
Denatured Protein ◽  
Sds Page ◽  
Receptor Fragment

AbstractGlucocorticoids and their tissue receptors are involved in many metabolic and developmental processes. Until now only two short fragments with a total length of 200 amino acids were known from the glucocorticoid receptor of the pig. Therefore we sequenced the main part (2.1 kb) of the porcine receptor. In addition, we subcloned a cDNA fragment of this sequence coding for 135 aa of the modulatory region in a pET expression vector. The protein fragment was expressed in E. coli as a his-tag fusion protein. In the SDS-PAGE, the crude E. coli extracts showed an enrichment of a 15 kDa protein which corresponds to the estimated molecular weight for the receptor fragment.After lysis and Ni-NTA affinity chromatography under denaturing conditions the protein was further purified either by dialysis (native protein) or by SDS-PAGE (linearized form). Both forms were emulsified together in adjuvant and used for rabbit immunization.The resulting antibodies were characterized by western blot analysis, immunoprecipitation, and additionally by immunohistochemistry. Western blot analysis confirmed the binding of the denatured protein by the antiserum and revealed a high binding affinity. Immunoprecipitation demonstrated that both the occupied and unoccupied forms of the receptor are detected. The specificity of the antiserum for pGCR was additionally demonstrated by immunohistochemistry.

Determination of Na(+)-K(+)-ATPase alpha- and beta-isoforms and kinetic properties in mammalian liver

1992 ◽  
Vol 262 (6) ◽  
pp. C1491-C1499 ◽  
Author(s):  
Y. Sun ◽  
W. J. Ball
Keyword(s):  
Western Blot ◽  
Rat Liver ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Polyclonal Antibodies ◽  
Liver Microsomes ◽  
Kinetic Properties ◽  
Rat Tissues ◽  
Rat Liver Microsomes ◽  
Kidney Enzyme

While Western blot analysis clearly revealed the presence of the alpha- and beta-subunits of Na(+)-K(+)-ATPase in a variety of rat tissues, beta was not readily detectable in liver. This observation was consistent with a previous report indicating that Na(+)-K(+)-ATPase immunoprecipitated from rat liver gives no clear evidence for the presence of a beta-subunit (Hubert et al. Biochemistry 25: 4156-4163, 1986). However, Western blot analysis of density gradient-purified lamb and rat liver microsomes showed the presence of a protein with an approximate molecular mass of 42 kDa that was immunoreactive with beta-specific polyclonal antibodies as well as beta-directed monoclonal antibodies. Deglycosylation of this protein by N-glycosidase F generated a core protein (beta c, M(r) approximately 32,000) that had the identical electrophoretic mobility as the beta c protein of the purified kidney enzyme. Isoform-specific monoclonal and synthetic peptide-directed polyclonal antibodies were used to demonstrate the presence of only the alpha 1- and beta 1-proteins in the liver and the presence of beta 2 in rat brain. Functional studies then showed that although both rat and lamb liver enzymes had sensitivities to cardiac glycoside inhibition similar to that of their corresponding kidney enzyme, the lamb liver enzyme had higher affinities for Na+, K+, and ATP than the kidney enzyme.

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