iBlot2--CHEM 584 v1

D6 and DARC are decoy chemokine binding receptors. They had been studied extensively in recent years for their role in breast cancer studies. The purpose of this study was to improve some critical steps in Western Blot in order to detect and identify recombinant D6 and DARC which has been expressed intracellularly by Pichia-GS115. Total proteins of Pichia-GS115 were obtained by breaking the yeast cells using acid-washed glass beads. Then, the lysates were treated with treatment buffer before electrophoresed on SDS-polyacrylamide gel. Western Blot was carried out after SDS PAGE to detect and identify the presence of recombinant D6 and DARC. Several parameters of Western Blot were studied and improved to enhance the specificity of results obtained. It was found that blocking of nitrocellulose membrane with 3% (w/v) skimmed milk at room temperature for 1 hour, incubation of primary antibody at the dilution of 1: 300 at room temperature for 16 hours and incubation of secondary antibody at the dilution of 1: 1000 at room temperature for 1 hour were able to enhance the sensitivity and specificity of Western Blot results. With the aid of prestained protein ladder as a marker, recombinant D6 and also DARC were shown as clear and precise bands at the range of molecular weight of 50-60 kDa on developed films using ECL substrate. The film exposure time was found to be 5 minutes for D6 and 10 minutes for DARC. As a result, the studies showed that improved Western Blot condition detected and identified the presence of recombinant D6 and DARC in the cell pellets of Pichia-GS115

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Dirofilaria immitis proteins recognized by antibodies from individuals living with microfilaremic dogs

The Journal of Infection in Developing Countries ◽

10.3855/jidc.12711 ◽

2020 ◽

Vol 14 (12) ◽

pp. 1442-1447

Author(s):

Oswaldo M Torres-Chable ◽

Ligia G Brito-Argaez ◽

Ignacio R Islas-Flores ◽

Claudia V Zaragoza-Vera ◽

Maritza Zaragoza-Vera ◽

...

Keyword(s):

Mass Spectrometry ◽

Western Blot ◽

Human Serum ◽

Dirofilaria Immitis ◽

Nitrocellulose Membrane ◽

Total Proteins ◽

Serum Samples ◽

Sds Page ◽

The World ◽

Specific Proteins

Introduction: Dirofilaria immitis is a nematode that affects human health in several countries of the world. This study was conducted to examine whether serum samples from the owners of microfilaremic dogs present immunoreactivity to parasite proteins. Methodology: Eight serum samples from the owners of microfilaremic dogs were examined. Total proteins were extracted from adult worms and 12% SDS-PAGE was performed. The gel was electroblotted to a nitrocellulose membrane, and a Western blot (WB) was performed. Reactive bands of 22, 33, 39, 49, and 63 kDa in WB were excised from the gel and analyzed by mass spectrometry (MS). Results: The MS results showed the presence of 10 different proteins of D. immitis recognized by the human serum samples. Conclusions: These results indicate that in endemic areas of D. immitis, owners of infected dogs recognize specific proteins of the parasite, suggesting a possible infection.

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Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646610 ◽

1989 ◽

Vol 61 (03) ◽

pp. 437-441 ◽

Cited By ~ 28

Author(s):

Cindra Condra ◽

Elka Nutt ◽

Christopher J Petroski ◽

Ellen Simpson ◽

P A Friedman ◽

...

Keyword(s):

Molecular Weight ◽

Salivary Gland ◽

Amino Acid ◽

Amino Acid Sequence ◽

Western Blot ◽

Potent Inhibitor ◽

Factor Xa ◽

Coagulation Factor ◽

Sds Page ◽

Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

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Western Blot Analyses of Prekallikrein and its Activation Products in Human Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648157 ◽

1991 ◽

Vol 65 (04) ◽

pp. 382-388 ◽

Cited By ~ 9

Author(s):

Dulce Veloso ◽

Robert W Colman

Keyword(s):

Complex Formation ◽

Western Blot ◽

Human Plasma ◽

Contact System ◽

Plasma Activation ◽

Normal Plasma ◽

Sds Page ◽

Activation Products ◽

Major Antigen ◽

Formation Of Complexes

SummaryPrekallikrein (PK), a zymogen of the contact system, and its activation products, kallikrein (KAL), KAl-inhibitor complexes and fragments containing KAL epitope(s) have been detected in human plasma by immunoblotting with a monoclonal anti-human plasma PK antibody, MAb 13G1L. Detection of antigen-MAb 13G11 complexes with peroxidase-conjugated anti-IgG showed that the two variants of PK (85- and 88-kDa) are the only major antigen species in normal, non-activated plasma. Upon plasma activation with kaolin, the intensity of the PK bands decreased with formation of complexes of KAL with CL inhibitor (C1 INH) and α2-rrtzcroglobulin (α2M) identical to those formed by the purified proteins. Immunoblots of normal plasma showed good correlation between the PK detected and the amount of plasma assayed. Increasing amounts of KAL incubated with a constant volume of PK-deficient plasma showed increasing amounts of KAL and of KAL-C1 INH and KAL-α2M complexes. Complexes of KALantithrombin III (ATIII) and the ratio of KALα2M/ KAL-CL INH were higher in activated CL INH-deficient plasmas than in activated normal plasmas. Protein resolution by 3-12% gradient SDS-PAGE and epitope detection with [125I]MAb 13G11 showed four KALα2M species and a 45-kDa fragment(s) in both surface-activated normal plasma and complexes formed by purified KAL and α2M. Immunoblots of activated plasma also showed bands at the position of KALCL INH and KALATIII complexes. When α1-antitrypsin Pittsburgh (cα1-AT, Pitts) was added to plasma before activation, KAL-α1-ALPitts was the main complex. The non-activated normal plasma revealed only an overloaded PK band. This is the first report of an antibody that recognizes KAL epitope(s) in KAL-α2M, KALATIII and KALa1-α1Pitts complexes and in the 45-kDa fragment(s). Therefore, MAb 13G11 should be useful for studying the structure of these complexes as well as the mechanism of complex formation. In addition, immunoblotting with MAb 13G11 would allow detection of KAl-inhibitor complexes in patient plasmas as indicators of activation of the contact system.

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OP0054 NEW ROLE FOR PROTEINASE 3 IN IL-16 BIOACTIVITY CONTROL IN GRANULOMATOSIS WITH POLYANGIITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.1314 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 28.1-29

Author(s):

A. Kerstein-Staehle ◽

C. Alarcin ◽

J. Luo ◽

G. Riemekasten ◽

P. Lamprecht ◽

...

Keyword(s):

Cell Death ◽

Western Blot ◽

Disease Activity ◽

Chronic Inflammation ◽

Immune Cells ◽

Peripheral Blood ◽

Granulomatosis With Polyangiitis ◽

Granulomatous Inflammation ◽

Proteinase 3 ◽

Sds Page

Background:The immunomodulatory cytokine IL-16 is increased in several inflammatory and autoimmune diseases1. IL-16 recruits and activates CD4+ immune cells such as T cells, dendritic cells, or monocytes. IL-16 is produced by various immune and non-immune cells, but synthesis and storage of IL-16 is regulated differentially depending on the cell type and stimulation. For its biological activity, IL-16 cleavage by caspase-3 is required1. Necrotizing granulomatous inflammation is a hallmark of granulomatosis with polyangiitis (GPA) with neutrophil dysregulation as a central driver of chronic inflammation and autoimmunity2. Earlier studies showed a correlation between increased serum IL-16 and disease parameters in AAV, including GPA3, but functional evidence for a direct link between IL-16 and neutrophils in granulomatous inflammation is missing so far.Objectives:In this study we aim to identify a functional link between increased IL-16, neutrophils, and the autoantigen proteinase 3 (PR3) with regard to chronic inflammation and autoimmunity in GPA.Methods:IL-16 was measured in sera of GPA patients (n = 40) and healthy controls (HC, n = 50) by ELISA and correlated with clinical features, such as disease activity (BVAS), creatinine, GFR, VDI and PR3-ANCA status. IL-16 protein expression was analyzed in peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMN) from GPA patients and HC (n = 5, each) by SDS-PAGE and western blot. Binding affinity of recombinant pro-IL-16 to native human PR3 was assessed by microscale thermophoresis. Cleavage of pro-IL-16 by active human PR3 was performed at various time points at 37°C. Cleavage products were analyzed by SDS-PAGE and western blot.Results:Circulating IL-16 was significantly increased in GPA patients compared to HC. Elevated IL-16 positively correlated with BVAS, creatinine, VDI and PR3-ANCA status and negatively correlated with GFR. In PMBC and PMN from GPA and HC we identified different expression patters of precursor and active forms of IL-16. In healthy PBMC we found high amounts of precursor (80kD), pro-IL-16 (55kD) and active IL-16 (17kD). In contrast, PBMC from GPA patients had lower amounts of pro-IL-16 and no active IL-16, indicating activation and secretion of IL-16 due to inflammatory stimulation, as shown earlier5. In GPA PMN we detected no precursor IL-16, but pro-IL-16 and its active form, in contrast to very low amounts of all IL-16 forms in healthy PMN. Processing and release of IL-16 in neutrophils has been linked to apoptosis and secondary necrosis5. By interaction studies we demonstrated direct binding of pro-IL-16 to PR3 with a Kd of 10 nM. In a subsequent cleavage assay we confirmed IL-16 processing by PR3 in a time-dependent manner.Conclusion:Correlation of serum IL-16 with clinical features of GPA suggests that IL-16 is associated with markers of disease activity, tissue damage and autoreactivity. We showed that PBMC and PMN represent a source of IL-16 in GPA. By the identification of PR3 as an additional IL-16-activating enzyme we could demonstrate a potential link between excessive PR3 expression, cell death and IL-16-dependent mechanisms, contributing to chronic granulomatous inflammation and autoimmunity in GPA.References:[1]Glass, W. G. et al. Not-so-sweet sixteen: The role of IL-16 in infectious and immune-mediated inflammatory diseases. J. Interf. Cytokine Res. 26, 511–520 (2006).[2]Millet, A. et al. Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis. J. Clin. Invest. 125, 4107–4121 (2015).[3]Yoon, T. et al. Serum interleukin-16 significantly correlates with the Vasculitis Damage Index in antineutrophil cytoplasmic antibody-associated vasculitis. Arthritis Res. Ther. 22, 1–6 (2020).[4]Elssner, A. et al. IL-16 Is Constitutively Present in Peripheral Blood Monocytes and Spontaneously Released During Apoptosis. J. Immunol. 172, 7721–7725 (2004).[5]Roth, S. et al. Secondary necrotic neutrophils release interleukin-16C and macrophage migration inhibitory factor from stores in the cytosol. Cell Death Discov. 1, 15056 (2015).Disclosure of Interests:None declared

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AB0050 A NOVEL METHOD FOR ISOLATION OF EXOSOMES FROM SYNOVIAL FLUID

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.1996 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 1057.2-1057

Author(s):

Y. Liu ◽

Y. Huang ◽

Q. Huang ◽

S. Sun ◽

Z. Ji ◽

...

Keyword(s):

Synovial Fluid ◽

Western Blot ◽

Biological Fluid ◽

High Sensitivity ◽

Protein Distribution ◽

Sds Page ◽

Close Relationship ◽

Novel Method ◽

Low Levels

Background:Exosomes in synovial fluid (SF) has a close relationship with the pathogenesis of rheumatiod arthritis. As a complex biological fluid, SF presents challenges for exosomes isolation using standard methods, such as ExoquickTM kit and ultracentrifugation.Objectives:The study aims to compared the quality of exosomes separated by ExoquickTM kit (TM), ExoquickTM kit+ExoquickTC kit (TM-TC), ultracentrifugation (UC) and TM-TC+UC(TM-TC-UC) from SF.Methods:Exosomes was separated by TM, TM-TC, UC and TM-TC-UC respectively. The size and concentrations of exosomes were detected by high sensitivity flow cytometry for nanoparticle analysis. Total protein and RNA were extracted from exosomes. SDS-PAGE was used to detect the protein distribution of exosomes. Western blot was used to examine the level of albumin and exosomes marker (TSG101 and CD81).Results:There was no statistic difference in the diameters of exosomes separated by the four methods. The concentrations of exosomes in TM, TM-TC, TM-TC-UC and UC were (5.65±0.93), (3.02±1.19), (1.67±0.25) and (4.61±0.73) *109Particles/mL. The protein concentrations of exosomes separated by the four methods were consistent with the concentrations of exosomes. SDS-PAGE showed that the protein distribution of exosomes separated by the four methods were different. Low levels of albumin were detected in TM-TC and TM-TC-UC, while high levels of albumin in TM and UC. Total RNA concentrations from exosomes in TM-TC was higher than other groups.Conclusion:TM-TC can be used to obtain higher quality exosomes from SF for the study of exosome-enriched components.References:[1]Helwa I, et al, A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents. PloS one, 2017. 12(1): p. e0170628-e0170628.Figure 1.A: SDS-PAGE showed the protein distribution of exosomes; B: the detection of albumin, TSG101 and CD81 by western blot.Disclosure of Interests:None declared

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Production and characterization of monoclonal antibodies to the edta extract of Leptospira interrogans, serovar icterohaemorrhagiae

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86821996000500012 ◽

1996 ◽

Vol 29 (5) ◽

pp. 483-489

Author(s):

Lilian Terezinha de Queiroz Leite ◽

Mauricio Resende ◽

Wanderley de Souza ◽

Elizabeth R.S. Camargos ◽

Matilde Cota Koury

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Western Blot ◽

Silver Staining ◽

Colloidal Gold ◽

Leptospira Interrogans ◽

Outer Envelope ◽

Lethal Challenge ◽

Sds Page

Monoclonal antibodies (MABs) ivere produced against an etbylenediaminetetraacetate (EDTA) extract of Leptospira interrogans serovar icterohaemorrhagiae being characterized by gel precipitation as IgM and IgG (IgGl and IgG2b). The EDTA extract was detected as several bands by silver staining in SDS-PAGE. In the Western blot the bands around 20 KDa reacted with a monoclonal antibody, 47B4D6, and was oxidized by periodate and was not digested by pronase, suggesting that the determinant is of carbohydrate nature, lmmunocytochemistry, using colloidal gold labeling, showed that an EDTA extract determinant recognized by monoclonal antibody 47B4D6, is localized under the outer envelope of serovar icterohaemorrhagiae. Hoe AIAB raised against the EDTA extract was not able to protect hamsters from lethal challenge with virulent homologous leptospires.

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Identification of the German Cockroach Allergens in Korean Atopy Using SDS - PAGE and Western Blot Analysis

Annals of Dermatology ◽

10.5021/ad.2000.12.4.247 ◽

2000 ◽

Vol 12 (4) ◽

pp. 247

Author(s):

Chun Wook Park ◽

Sang Dong Kim ◽

Cheol Heon Lee ◽

Dong-Kyu Lee

Keyword(s):

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

German Cockroach ◽

Sds Page

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Σύνθεση και μελέτη της δομής και της βιοδραστικότητας οργανοκασσιτερικών ενώσεων με αρυλιμινο υποκατεστημένα βενζοϊκά οξέα

10.12681/eadd/36060 ◽

2015 ◽

Author(s):

Muvudji Cephas Mwanasaka

Keyword(s):

Western Blot ◽

1H Nmr ◽

13C Nmr ◽

Sds Page ◽

Ft Ir

Στα πλαίσια της παρούσας διδακτορικής διατριβής πραγματοποιήθηκε μια διεπιστημονική ερευνητική εργασία, η οποία είχε ως κεντρική ιδέα τη χημεία και τη βιοδραστικότητα των οργανοκασσιτερικών ενώσεων. Για την επίτευξη της μελέτης αυτής, η ερευνητική εργασία κινήθηκε σε τρεις κεντρικούς άξονες: σχεδιασμός και σύνθεση, ανάλυση και ταυτοποίηση και, τέλος, in vitro μελέτη βιολογικών δράσεων. Αρχικά σχεδιάστηκαν και συντέθηκαν ιμίνες (βάσεις Schiff) παράγωγα του βενζοϊκού οξέος, από τη συμπύκνωση του 3- και του 4-αμινοβενζοϊκου οξέος με π-υποκατεστημένες (Η, ΝΟ2, Cl, MeO, Me2N) βενζαλδεΰδες, καθώς και από τη συμπύκνωση των π-υποκατεστημένων (Η, ΝΟ2, Cl, MeO, Me2N) ανιλίνων με π-φορμυλοβενζοϊκό οξύ. Από τις αντιδράσεις αυτές συντέθηκαν βιολογικά ενεργά αρυλιμηνο υποκατεστημένα βενζοϊκά οξέα, τα οποία χρησιμοποιήθηκαν στη συνέχεια της συνθετικής διαδικασίας ως ligands (L) για τη σύνθεση νέων τριοργανοκασσιτερικών εστέρων γενικού τύπου R3SnL με R= Μe και Ph. Η ταυτοποίηση και ο χαρακτηρισμός των νέων αυτών ligands και των συμπλόκων που συντέθηκαν πραγματοποιήθηκε με συνδυασμό αναλυτικών τεχνικών: φασματοσκοπικές τεχνικές (FT-IR, 1H-NMR, 13C-NMR, UV-vis), στοιχειακή ανάλυση και την κρυσταλλογραφία ακτίνων Χ. Επόμενο στάδιο ήταν η in vitro μελέτη βιοδραστικότητας των ενώσεων αυτών η οποία, χωρίστηκε σε δύο σκέλη. Το ένα βιολογικό σκέλος κινήθηκε σε κυτταρικό επίπεδο, όπου αξιολογήθηκε πρώτα η in vitro αποπτωτική δράση σε τέσσερεις καρκινικές κυτταρικές σειρές, ΗepG2, HeLa, K562 και N2a. Για το σκοπό αυτό χρησιμοποιήθηκε η δοκιμή ΜΤΤ (ΜΤΤ assay), η οποία εκτιμά την επίδραση στη βιωσιμότητα των κυττάρων μετά από έκθεσή τους σε διάφορες συγκεντρώσεις των ουσιών. Στη συνέχεια έγιναν νευροκυτταροτοξικές μελέτες, εκτιμώντας την ενδεχόμενη in vitro αναπτυξιακή νευροτοξικότητα. Για το σκοπό αυτό αξιολογήθηκε η επίδραση των υπό μελέτη ουσιών στη διαδικασία ανάπτυξης αξονικών προεκβολών σε υπό διαφοροποίηση Ν2a κύτταρα, μετά από έκθεση σε υποκυτταροτοξικές συγκεντρώσεις των υπό μελέτη ουσιών. Το άλλο βιολογικό σκέλος κινήθηκε σε μοριακό επίπεδο, στοχεύοντας στην εκτίμηση της επίδρασης των υπό μελέτη ουσιών σε συγκεκριμένους βιοχημικούς παράγοντες με σκοπό τη διαλεύκανση των βιοχημικών μηχανισμών δράσεών τους. Εκτιμήθηκε η δράση τους σε συγκεκριμένες πρωτεΐνες του νευροκυτταρικού σκελετού, χρησιμοποιώντας SDS-PAGE και ανοσοχημική τεχνική ανάλυσης Western blot. Επιπλέον, έγινε εκτίμηση της in vitro ανασταλτικής δράσης τους στη λιποξυγονάση, ένζυμο που είναι γνωστό για το ρόλο του στην παραγωγή ελευθέρων ριζών στη φλεγμονή. Τέλος, εκτιμήθηκε η ενδεχόμενη in vitro αντιοξειδωτική δράση τους, αξιολογώντας την ικανότητά τους στην αναστολή της επαγόμενης από το ΑΑΡΗ λιπιδικής υπεροξείδωσης, καθώς και την ικανότητα αλληλεπίδρασής τους με τη σταθερή ρίζα DPPH. Από τα αποτελέσματα που προέκυψαν προσπαθήσαμε να βγάλουμε συμπεράσματα για το κατά πόσο η δομή των καρβοξυλικών οξέων που χρησιμοποιήθηκαν ως Ligands επηρεάζει τόσο τον τρόπο συναρμογής της καρβοξυλικής ομάδας με το τριοργανοκασσιτερικό κατιόν, όσο και τη βιολογική δράση των υπό μελέτη τριοργανοκασσιτερικών εστέρων στις προαναφερόμενες μελέτες.

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