scholarly journals The Effect of Breed, Gender, and Acid Stimulation in Dog Saliva Proteome

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Sónia Lucena ◽  
Ana V. Coelho ◽  
Fernando Capela-Silva ◽  
Asta Tvarijonaviciute ◽  
Elsa Lamy
Keyword(s):  
Gel Electrophoresis ◽  
Protein Concentration ◽  
Animal Species ◽  
Lemon Juice ◽  
Minimal Effect ◽  
Total Protein Concentration ◽  
Two Dimensional Gel Electrophoresis ◽  
Sds Page ◽  
Dog Breed ◽  
Purebred Dogs

Saliva gained interest as a potential noninvasive source of biomarkers in humans and that interest starts to be extended also to other animal species. For this purpose, the knowledge of the salivary proteome in healthy conditions and the factors that affect it and how they affect it are necessary. The aim of the present study was to assess the effect that gender and breed have in saliva proteome and the changes in it induced by stimulation with acid. Saliva from 4 different purebred dogs (Portuguese Podengo, Greyhound, Rafeiro Alentejano, and Beagle) of both genders was collected without and after stimulation with lemon juice. SDS-PAGE and two-dimensional gel electrophoresis (2-DE) profiles were compared and the proteins of interest in-gel digested and identified by mass spectrometry. Acid stimulation decreased total protein concentration and the relative amounts of some protein bands/spots. Gender appeared to have minimal effect in saliva proteome, whereas the influence of breed varies. Beagles and Portuguese Podengos were the two breeds with higher differences. In conclusion, stimulation procedures and dog breed should be considered in data analysis when using salivary proteins for diagnostic purposes.

Thermal gelation and denaturation of bovine β-lactoglobulins A and B

1994 ◽  
Vol 61 (2) ◽  
pp. 221-232 ◽  
Author(s):  
Mary McSwiney ◽  
Harjinder Singh ◽  
Osvaldo Campanella ◽  
Lawrence K. Creamer
Keyword(s):  
Heat Treatment ◽  
Rheological Properties ◽  
Functional Property ◽  
Gel Electrophoresis ◽  
Protein Concentration ◽  
Gel Formation ◽  
Native Page ◽  
Thermal Gelation ◽  
Sds Page ◽  
Important Functional Property

SummaryHeat-induced gelation, an important functional property of β-lactoglobulin, was studied by measuring the rheological properties of both the A and B variants of the protein during and after heat treatment within a range of pH, temperature and concentration. Gel electrophoresis was used to determine the extent of denaturation and disulphide bond crosslinking of some samples. Both variants formed gel networks on heating at temperatures > 75 °C, and under most conditions the storage modulus (G′) of βlactoglobulin A gels was higher than the G′ of β-lactoglobulin B gels, in particular after cooling to 25 °C. A minimum protein concentration of 50 g/1 was required for gel formation at pH 7·0 in 0·1 M-NaCl by both variants at 80 °C. Increasing the protein concentration above 50 g/1 increased G′, the extent of increase being much greater for the A variant than the B variant. G′ of variant A gels was not much influenced by pH whereas G′ of variant B gels decreased slightly from pH 3 to pH 6 and increased between pH 6 and pH 9. When mixtures of the two variants were gelled G′ increased at the temperature of heating (80 °C) and after cooling (25 °C) as the relative quantity of variant A was increased. Comparisons of the loss of discrete protein bands from electrophoretic gels (native-PAGE, SDS-PAGE and SDS-PAGE of reduced samples) showed that heating β-lactoglobulin solutions of 100 g/1 at pH 7 in 0·1 M-NaCl and at 75, 80 and 85 °C caused a faster loss of both native and SDS-soluble β-lactoglobulin A than of β-lactoglobulin B. It was concluded that the loss of native β-lactoglobulin structure from these solutions during heating was faster than the formation of disulphidelinked aggregates, which was faster than gel formation for both β-lactoglobulin A and β-lactoglobulin B, and that each of these reactions was faster for β-lactoglobulin A than for β-lactoglobulin B. This contrasts with conclusions drawn from some previous studies and may arise from the differences in protein concentration between the present study (∼ 100 g/1) and the previous ones (< ∼ 10 g/1).

scholarly journals Two-dimensional gel electrophoresis and immunoblotting for detection of antigenic proteins from natural rubber latex Gel elektroforesis 2-dimensi dan imunobloting untuk deteksi protein antigen dari lateks karet alam

2016 ◽  
Vol 80 (2) ◽  
Author(s):  
. Siswanto
Keyword(s):  
New Zealand ◽  
Natural Rubber ◽  
Gel Electrophoresis ◽  
Natural Rubber Latex ◽  
Rubber Latex ◽  
Two Dimensional Gel Electrophoresis ◽  
Antigenic Protein ◽  
Antigenic Proteins ◽  
Rabbit Igg ◽  
Sds Page

AbstrakLateks karet alam banyak digunakan untuk produksi peralatan medis, industri dan rumah tangga. Reaksi alergi yang disebabkan oleh protein asal lateks telah banyak dilaporkan terutama berkaitan dengan penggunaan sarung tangan asal karet alam. Namun tidak semua jenis protein dari karet alam bisamenyebabkan alergi. Penelitian ini dilakukan untuk mendeteksi jenis protein antigenik yang berasal dari karet alam menggunakan teknik imunobloting. Protein diekstrak dari tiga fraksi sentrifugasi lateks (serum B sebagai fraksi dasar, serum C atau serum sitosolik sebagai fase tengah dan partikel karet sebagai fase atas) dan tujuh jenis sarung tangan komersial, kemudian dipisahkan berdasarkan berat molekulnya melalui Gel elektroforesis 1-D (SDS PAGE) dan 2-D. Selanjutnya untuk deteksi protein antigenik secara immuno-chemiluminescense dilakukan imunobloting menggunakan IgG antibodi poliklonal anti protein lateks dari kelinci putih New Zealand dan diwarnai dengan Sypro Ruby protein blot fluorescence. Hasil imunobloting menunjukkan bahwa tidak semua protein serum C dan serum B bersifat antigenik. Terdapat lebih dari 14 spot protein antigenik yang terdeteksi dari serum C pada posisi pI antara pH 4,2 s/d pH 6,8, serta lebih dari 16 spot protein antigenik yang terdeteksi pada serum B dengan pI antara pH 5,5 s/d pH 7,0. Protein yang bersifat antigenik dalam serum C a.l: dengan BM 43 kDa diduga Hev b 7,01, BM 22 kDa adalah Hev b 3 dan BM 15 kDa adalah Hev b 8. Sedangkan protein antigenik dalam serum B dengan BM 42 kDa diduga adalah Hev b 10, dan BM 39 kDa adalah Hev b 2. Protein yang bersifat antigenik pada sarung tangan yang terdeteksi dengan IgG kelinci anti serum-C antara lain Hev b 5 dengan BM 14 kDa, Hev b 1 (BM 10 kDa), Hev b 6.03 (BM 18 dan 19 kDa), dan Hev b9 (BM 55 kDa). Sedangkan yang terdeteksi dengan antibodi IgG anti serum B antara lain Hev b 6.02 dengan BM 7 kDa, serta Hev b 10 (BM 46 kDa) dan Hev b9 (BM 55 kDa). AbstractNatural rubber latex is widely used for the production of medical, industrial and household devices. Allergic reactions caused by latex proteins have been reported primarily related with the use of natural rubber gloves. However, not all types of proteins from natural rubber can cause allergies. This study was conducted to detect the type of antigenic proteins derived from natural rubber with immunobloting techniques. Proteins were extracted from three fractions of latex centrifugation (Bserum as bottom fractions, C-serum or cytosolic serum asmiddle phase and rubber particles as upper phase) and seven types of commercial gloves, then separated by molecular weight through 1-D gel electrophoresis (SDS PAGE) and 2-D. Detection of antigenic protein byimmuno-chemiluminescense, was performed by immunoblotting using polyclonal antibody IgG anti-protein latex from New Zealand white rabbits and stained withSypro Ruby protein blot fluorescence. Immunobloting results indicate that not all proteins from C-serum and Bserum were antigenic. More than 14 antigenic protein spots were detected in the samples of C-serum at pI between pH 4.2 to pH 6.8, and more than 16 antigenic protein spots were detected in the samples of B-serum at pI between pH 5.5 to pH 7.0. Antigenic proteins detected in C-serum were MW 43 kDa suspected as Hev b 7:01, MW 22 kDa was Hev b 3 and MW 15 kDa was Hev b 8. While the antigenic protein detected in B-serumwith MW 42 kDa was suspected as Hev b 10, and protein with MW 39 kDa was Hev b 2. Antigenic proteins detected on the rubber gloves with rabbit IgG anti-Cserum were Hev b 5 with MW 14 kDa, Hev b 1 (MW 10 kDa), Hev b 6:03 (MW 18 and 19 kDa), and Hev b9 (MW 55 kDa). Whereas antigenic protein of rubbergloves detected with IgG anti-B serum were Hev b 6:02 with MW 7 kDa, and Hev b 10 (46 kDa) and Hev b9 (MW 55 kDa).

scholarly journals Defining E-cadherin-associated protein complexes in epithelial cells: plakoglobin, beta- and gamma-catenin are distinct components

1993 ◽  
Vol 104 (3) ◽  
pp. 751-762 ◽  
Author(s):  
P.A. Piepenhagen ◽  
W.J. Nelson
Keyword(s):  
Gel Electrophoresis ◽  
Mdck Cells ◽  
Apparent Molecular Weight ◽  
Beta Catenin ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Sds Page ◽  
Alpha Beta ◽  
The Relationship ◽  
E Cadherin

Ca(2+)-dependent cell adhesion is mediated by a family of proteins termed cadherins, and is modulated by cytosolic proteins that include alpha-, beta-, and gamma-catenin and other cytoskeletal proteins that bind to the cytoplasmic domain of cadherins. Recent studies have suggested that either beta- or gamma-catenin may be identical to plakoglobin, a protein associated with adherens junctions. However, the relationship between these proteins, and their interaction with cadherins, are not well understood. In this study, we have further defined the relationship between plakoglobin and the catenins in complexes with E-cadherin in Madin-Darby canine kidney (MDCK) cells. Specific immunoprecipitations revealed that plakoglobin (86 kDa) and beta-catenin (92 kDa) have different detergent extractabilities and apparent molecular weights in these cells; however, plakoglobin has an apparent molecular weight similar to that of gamma-catenin (86 kDa). Immunoblotting of E-cadherin immunoprecipitates demonstrated that both plakoglobin and beta-catenin co-immunoprecipitate with E-cadherin. Laser-scanning confocal microscopy demonstrated temporally and spatially co-ordinate redistribution of plakoglobin and E-cadherin following induction of cell-cell contact in MDCK cells. Although plakoglobin comigrated with gamma-catenin on SDS-PAGE, quantitative analysis of E-cadherin and plakoglobin immunoprecipitates revealed that plakoglobin accounted for &lt; 50% of the gamma-catenin signal. Two-dimensional gel electrophoresis resolved the gamma-catenin protein band into two proteins. One protein was identified as plakoglobin, based upon apparent molecular weight, immunoreactivity and isoelectric point (pI approximately 6.1). The other protein comigrated with gamma-catenin on SDS-PAGE, did not react with plakoglobin antibodies and had a pI of approximately 4.25; we refer to this protein as gamma-catenin to distinguish it from plakoglobin. Two-dimensional gel electrophoresis further revealed that plakoglobin comprised multiple isoelectric variants, but that, within the newly synthesized pool of plakoglobin, only the most basic of these variants co-immunoprecipitated with E-cadherin; phosphorylation did not account for the plakoglobin isoelectric variants seen by two-dimensional gel electrophoresis. These results demonstrate directly that plakoglobin associates and co-localizes with the E-cadherin in MDCK epithelial cells in a complex that contains alpha-, beta-, and gamma-catenin. Although plakoglobin shares sequence similarity with beta-catenin, and comigrates with gamma-catenin in SDS-PAGE, plakoglobin is distinct from the catenins. The association of plakoglobin with E-cadherin may be regulated by post-translational modifications of plakoglobin.

scholarly journals Urinary Proteomics Reveals Promising Biomarkers in Menstrually Related and Post-Menopause Migraine

2021 ◽  
Vol 10 (9) ◽  
pp. 1854
Author(s):  
Elisa Bellei ◽  
Stefania Bergamini ◽  
Cecilia Rustichelli ◽  
Emanuela Monari ◽  
Michele Dal Porto ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Large Scale ◽  
World Population ◽  
Vascular Disorder ◽  
Liquid Chromatography Mass Spectrometry ◽  
Urinary Proteins ◽  
Two Dimensional Gel Electrophoresis ◽  
Apolipoprotein A ◽  
Sds Page ◽  
Post Menopause

Migraine is an invalidating neuro-vascular disorder largely spread in the world population. Currently, its pathophysiology is not yet completely understood. The purpose of this study was to investigate the urinary proteome of women suffering from menstrually related migraine (MM) and post-menopause migraine (PM) in comparison with non-headache women as controls, to search potential biomarkers of these migraine sub-types. Urine samples were analyzed by mono-dimensional gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2DE) coupled to liquid chromatography-mass spectrometry (LC-MS/MS). Twenty-one urinary proteins were found significantly dysregulated in MM and PM (p < 0.05). The STRING Analysis database revealed interaction between 15 proteins, which were mainly involved in the immune and inflammatory response. Seven of the most considerable proteins were further quantified by western blot: protein S100A8 (S10A8), up-regulated in MM, uromodulin (UROM), alpha-1-microglobulin (AMBP), gelsolin (GELS), prostaglandin-H2 D-isomerase (PTGDS), over-expressed in PM, apolipoprotein A-I (APOA1), and transthyretin (TTHY), respectively down- and up-regulated in both migraineur groups vs controls. These candidate biomarkers might be involved in the neurophysiological network of MM and PM, thus helping to better understand the pathophysiology of these migraine forms. If validated in large-scale studies, this protein cluster could become a distinctive target for clinical applications in migraine diagnosis and treatment.

scholarly journals Two-dimensional gel electrophoresis and immunoblotting for detection of antigenic proteins from natural rubber latex Gel elektroforesis 2-dimensi dan imunobloting untuk deteksi protein antigen dari lateks karet alam

2016 ◽  
Vol 80 (2) ◽  
Author(s):  
. Siswanto
Keyword(s):  
New Zealand ◽  
Natural Rubber ◽  
Gel Electrophoresis ◽  
Natural Rubber Latex ◽  
Rubber Latex ◽  
Two Dimensional Gel Electrophoresis ◽  
Antigenic Protein ◽  
Antigenic Proteins ◽  
Rabbit Igg ◽  
Sds Page

AbstrakLateks karet alam banyak digunakan untuk produksi peralatan medis, industri dan rumah tangga. Reaksi alergi yang disebabkan oleh protein asal lateks telah banyak dilaporkan terutama berkaitan dengan penggunaan sarung tangan asal karet alam. Namun tidak semua jenis protein dari karet alam bisamenyebabkan alergi. Penelitian ini dilakukan untuk mendeteksi jenis protein antigenik yang berasal dari karet alam menggunakan teknik imunobloting. Protein diekstrak dari tiga fraksi sentrifugasi lateks (serum B sebagai fraksi dasar, serum C atau serum sitosolik sebagai fase tengah dan partikel karet sebagai fase atas) dan tujuh jenis sarung tangan komersial, kemudian dipisahkan berdasarkan berat molekulnya melalui Gel elektroforesis 1-D (SDS PAGE) dan 2-D. Selanjutnya untuk deteksi protein antigenik secara immuno-chemiluminescense dilakukan imunobloting menggunakan IgG antibodi poliklonal anti protein lateks dari kelinci putih New Zealand dan diwarnai dengan Sypro Ruby protein blot fluorescence. Hasil imunobloting menunjukkan bahwa tidak semua protein serum C dan serum B bersifat antigenik. Terdapat lebih dari 14 spot protein antigenik yang terdeteksi dari serum C pada posisi pI antara pH 4,2 s/d pH 6,8, serta lebih dari 16 spot protein antigenik yang terdeteksi pada serum B dengan pI antara pH 5,5 s/d pH 7,0. Protein yang bersifat antigenik dalam serum C a.l: dengan BM 43 kDa diduga Hev b 7,01, BM 22 kDa adalah Hev b 3 dan BM 15 kDa adalah Hev b 8. Sedangkan protein antigenik dalam serum B dengan BM 42 kDa diduga adalah Hev b 10, dan BM 39 kDa adalah Hev b 2. Protein yang bersifat antigenik pada sarung tangan yang terdeteksi dengan IgG kelinci anti serum-C antara lain Hev b 5 dengan BM 14 kDa, Hev b 1 (BM 10 kDa), Hev b 6.03 (BM 18 dan 19 kDa), dan Hev b9 (BM 55 kDa). Sedangkan yang terdeteksi dengan antibodi IgG anti serum B antara lain Hev b 6.02 dengan BM 7 kDa, serta Hev b 10 (BM 46 kDa) dan Hev b9 (BM 55 kDa). AbstractNatural rubber latex is widely used for the production of medical, industrial and household devices. Allergic reactions caused by latex proteins have been reported primarily related with the use of natural rubber gloves. However, not all types of proteins from natural rubber can cause allergies. This study was conducted to detect the type of antigenic proteins derived from natural rubber with immunobloting techniques. Proteins were extracted from three fractions of latex centrifugation (Bserum as bottom fractions, C-serum or cytosolic serum asmiddle phase and rubber particles as upper phase) and seven types of commercial gloves, then separated by molecular weight through 1-D gel electrophoresis (SDS PAGE) and 2-D. Detection of antigenic protein byimmuno-chemiluminescense, was performed by immunoblotting using polyclonal antibody IgG anti-protein latex from New Zealand white rabbits and stained withSypro Ruby protein blot fluorescence. Immunobloting results indicate that not all proteins from C-serum and Bserum were antigenic. More than 14 antigenic protein spots were detected in the samples of C-serum at pI between pH 4.2 to pH 6.8, and more than 16 antigenic protein spots were detected in the samples of B-serum at pI between pH 5.5 to pH 7.0. Antigenic proteins detected in C-serum were MW 43 kDa suspected as Hev b 7:01, MW 22 kDa was Hev b 3 and MW 15 kDa was Hev b 8. While the antigenic protein detected in B-serumwith MW 42 kDa was suspected as Hev b 10, and protein with MW 39 kDa was Hev b 2. Antigenic proteins detected on the rubber gloves with rabbit IgG anti-Cserum were Hev b 5 with MW 14 kDa, Hev b 1 (MW 10 kDa), Hev b 6:03 (MW 18 and 19 kDa), and Hev b9 (MW 55 kDa). Whereas antigenic protein of rubbergloves detected with IgG anti-B serum were Hev b 6:02 with MW 7 kDa, and Hev b 10 (46 kDa) and Hev b9 (MW 55 kDa).

scholarly journals Expression of melanotransferrin isoforms in human serum: relevance to Alzheimer's disease

2003 ◽  
Vol 374 (2) ◽  
pp. 463-471 ◽  
Author(s):  
Richard R. DESROSIERS ◽  
Yanick BERTRAND ◽  
Quynh-Tran NGUYEN ◽  
Michel DEMEULE ◽  
Reinhard GABATHULER ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Human Serum ◽  
Gel Electrophoresis ◽  
Post Translational Modification ◽  
Two Dimensional Gel Electrophoresis ◽  
Control Subjects ◽  
Bivalent Cations ◽  
Sds Page ◽  
High Level

Levels of soluble melanotransferrin in serum have been reported to be higher in patients with Alzheimer's disease than in control subjects. The present study investigated melanotransferrin in human body fluids in the light of these findings. To clarify the correlation between melanotransferrin and Alzheimer's disease, the melanotransferrin content was determined by non-reducing, denaturing SDS/PAGE and Western blotting. Under these conditions, serum melanotransferrin migrated at 79 and 82 kDa. Melanotransferrin antigenicity and the relative proportions of the two forms were very sensitive to factors that altered its conformation, including disulphide bridges, pH and bivalent cations. Serum melanotransferrin levels were not significantly different between control subjects and patients with Alzheimer's disease using whole serum, EDTA-supplemented serum or serum immunoglobulin-depleted by Protein G–Sepharose and enriched by affinity precipitation with the lectin from Asparagus pea. Glycosylated forms of serum melanotransferrin bound to Asparagus lectin manifested similar patterns on two-dimensional gel electrophoresis in samples from controls and Alzheimer's disease subjects. Melanotransferrin was also present in saliva and at a high level in urine, but contents were similar in controls and patients with Alzheimer's disease. Together, these results demonstrate that serum melanotransferrin exists in various conformations depending on the binding of bivalent cations or following post-translational modification. These data also indicate that human serum melanotransferrin levels are unchanged in subjects with Alzheimer's disease.

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