scholarly journals Immunological comparison of the NADH:nitrate reductase from different cucumber tissues

2014 ◽  
Vol 64 (3) ◽  
pp. 245-250
Author(s):  
Jolanta Marciniak ◽  
Grażyna Kłobus ◽  
Józef Buczek ◽  
Tadeusz Stefaniak ◽  
Jarosław Mazur
Keyword(s):  
Nitrate Reductase ◽  
Cucumis Sativus ◽  
Polyclonal Antibodies ◽  
Specific Antibodies ◽  
Immunological Comparison ◽  
Cucumber Roots ◽  
Sepharose 4B ◽  
Blue Sepharose

Soluble nitrate reductase from cucumber roots (<i>Cucumis sativus</i> L.) was isolated and purified with blue-Sepharose 4B. Specific antibodies against the NR protein were raised by immunization of a goat. Using polyclonal antibodies anti-NR properties of the nitrate reductase from various cucumber tissues were examined. Experiments showed difference in immuno-logical properties of nitrate reductase (NR) from cotyledon roots and leaves.

Determination of Ochratoxin A in Feed by Immunoaffinity Cleanup and Liquid Chromatography

2013 ◽  
Vol 96 (3) ◽  
pp. 599-602 ◽  
Author(s):  
Ping Ding ◽  
Ziyou Mi ◽  
Yali Hou ◽  
Yigang He ◽  
Jianhua Xie
Keyword(s):  
Liquid Chromatography ◽  
Detection Limit ◽  
Ochratoxin A ◽  
Cyanogen Bromide ◽  
Polyclonal Antibodies ◽  
Immunoaffinity Column ◽  
Low Levels ◽  
Sepharose 4B ◽  
Feed Samples

Abstract A method using LC was developed for determination of ochratoxin A (OTA) in feeds. The extracted samples were cleaned up by an immunoaffinity column prepared by covalently coupling polyclonal antibodies against OTA to cyanogen bromide-activated Sepharose 4B. The eluates were determined by LC with fluorescence detection. Recoveries of OTA from fortified samples of 1–10 μg/kg levels ranged from 84.3 to 90.0%, with CVs of 3.3–7.8%. The detection limit was 0.045 μg/kg based on an S/N of 3:1. A total of 65 feed samples were screened for OTA with the proposed method. The results showed that only nine samples were contaminated with OTAs at low levels. The presented method was successfully applied to quantify OTAs in real feed samples.

Studies of Microthrix parvicella in situ and in laboratory culture: production and use of specific antibodies

2002 ◽  
Vol 46 (1-2) ◽  
pp. 115-118 ◽  
Author(s):  
N. Connery ◽  
A.S. Thompson ◽  
S. Patrick ◽  
M.J. Larkin
Keyword(s):  
Wastewater Treatment ◽  
Wastewater Treatment Plant ◽  
Doubling Time ◽  
Polyclonal Antibodies ◽  
Treatment Plant ◽  
Laboratory Culture ◽  
Specific Antibodies ◽  
Culture Production ◽  
Microthrix Parvicella

Physiological studies on M. parvicella have been conducted to determine the rate of growth of this organism in pure culture. The organism displayed a doubling time of 128 days despite its profuse abundance in a local Wastewater Treatment Plant (WWTW). An extensive survey has been ongoing since February 2000 into the extent of M. parvicella in the WWTW. A suite of monoclonal and polyclonal antibodies has been developed to detect and quantify M. parvicella.

scholarly journals A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin fromClostridium septicum

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3407 ◽  
Author(s):  
Lorena Vázquez-Iglesias ◽  
Borja Estefanell-Ucha ◽  
Leticia Barcia-Castro ◽  
María Páez de la Cadena ◽  
Paula Álvarez-Chaver ◽  
...  
Keyword(s):  
Protein Purification ◽  
Polyclonal Antibodies ◽  
Clinical Intervention ◽  
Farm Animals ◽  
Alpha Toxin ◽  
Slot Blot ◽  
Clostridium Septicum ◽  
Time Saving ◽  
Specific Antibodies

Clostridium septicumproduces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced byClostridium septicum.This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin ofClostridium septicumand produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot.

scholarly journals Porphyromonas gingivalis Fimbriae Bind to Cytokeratin of Epithelial Cells

2002 ◽  
Vol 70 (1) ◽  
pp. 96-101 ◽  
Author(s):  
Hakimuddin T. Sojar ◽  
Ashu Sharma ◽  
Robert J. Genco
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Porphyromonas Gingivalis ◽  
Polyclonal Antibodies ◽  
Cell Extract ◽  
Host Cells ◽  
Amino Terminal ◽  
A Minor ◽  
Sepharose 4B ◽  
Overlay Assay

ABSTRACT The adherence of Porphyromonas gingivalis to host cells is likely a prerequisite step in the pathogenesis of P. gingivalis-induced periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are shown to be involved in this process. Little is known regarding epithelial receptor(s) involved in binding of P. gingivalis fimbriae. Using an overlay assay with purified P. gingivalis fimbriae as a probe, two major epithelial cell proteins with masses of 50 and 40 kDa were identified by immunoblotting with fimbria-specific antibodies. Iodinated purified fimbriae also bound to the same two epithelial cell proteins. An affinity chromatography technique was utilized to isolate and purify the epithelial components to which P. gingivalis fimbriae bind. Purified fimbriae were coupled to CNBr-activated Sepharose-4B, and the solubilized epithelial cell extract proteins bound to the immobilized fimbriae were isolated from the column. A major 50-kDa component and a minor 40-kDa component were purified and could be digested with trypsin, suggesting that they were proteins. These affinity-eluted 50- and 40-kDa proteins were then subjected to amino-terminal sequencing, and no sequence could be determined, suggesting that these proteins have blocked amino-terminal residues. CNBr digestion of the 50-kDa component resulted in an internal sequence homologous to that of Keratin I molecules. Further evidence that P. gingivalis fimbriae bind to cytokeratin molecule(s) comes from studies showing that multicytokeratin rabbit polyclonal antibodies cross-react with the affinity-purified 50-kDa epithelial cell surface component. Also, binding of purified P. gingivalis fimbriae to epithelial components can be inhibited in an overlay assay by multicytokeratin rabbit polyclonal antibodies. Furthermore, we showed that biotinylated purified fimbriae bind to purified human epidermal keratin in an overlay assay. These studies suggest that the surface-accessible epithelial cytokeratins may act as receptor(s) for P. gingivalis fimbriae. We hypothesize that adherence of P. gingivalis fimbriae to cytokeratin may be important for colonization of oral mucous membranes and possibly also for activation of epithelial cells.

Nitrate Reductase from a Mutant Strain of Chlamydomonas reinhardii Incapable of Nitrate Assimilation

1983 ◽  
Vol 38 (5-6) ◽  
pp. 439-445 ◽  
Author(s):  
Emilio Fernández ◽  
Jacobo Cárdenas
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Wild Strain ◽  
Nitrate Assimilation ◽  
Pyridine Nucleotide ◽  
Spectral Studies ◽  
Binding Region ◽  
Chlamydomonas Reinhardii ◽  
Blue Sepharose

Nitrate reductase from mutant 305 of Chlamydomonas reinhardii has been purified about 90-fold and biochemically characterized. The enzyme can use reduced flavins and viologens as electron donors to reduce nitrate but, unlike the nitrate reductase complex from its parental wild strain, lacks NAD(P)H-nitrate reductase and NAD(P)H-cytochrome c reductase activities, does not bind to Blue-Agarose or Blue-Sepharose and exhibits a significantly lower molecular weight (177.000 vs. 241.000), whereas its kinetic characteristics and its sensitivity against several inhibitors and treatments are very similar to those of the terminal nitrate reductase activity of the wild strain complex. Spectral studies and antagonistic experiments with tungstate show the presence of cytochrome b557 and molybdenum. These facts lead us to propose that nitrate reductase from mutant 305 has a protein deletion which affects the pyridine nucleotide binding region of the diaphorase protein but without any effect on the terminal nitrate reductase activity.

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