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| − | + | Next, H-ficolin (200 nM) in running buffer was injected over the GlcNAc-immobilized chip for 100 s (association) followed by buffer flow for 200 s (dissociation). To characterize the binding of nIgG peptides to H-ficolin, separate second injections of increasing concentrations of nIgG peptides were made under similar running conditions. During the experiment, the flow rate was constantly maintained at 30 ml/min. The bound proteins were removed after one cycle by injecting 15 ml of 0.1 M NaOH (regeneration buffer). We used specific buffers for simulating the ``normal condition'' with TBS buffer (25 mM Tris, 145 mM NaCl, pH 7.4, 2.5 mM CaCl2) and the ``infection-inflammation condition'' with MBS buffer (25 mM MES, 145 mM NaCl, pH 6.5, 2 mM CaCl2). The SPR sensograms were analyzed by the BIAevaluation 3.2 software and the KD (dissociation constant) was calculated using 151 Langmuir binding model. The plots were finally made by overlaying the original binding curves (black) with the 151 binding model fitted curves (red). Controls used for normalization were obtained by injection of buffers alone instead of the proteins. The difference in the value of the resonance unit before and after injection is a measure of the peptide:protein interaction. The plots shown are representative of three independent experiments. Statistical analysis. For all the experiments, three replicates were performed per sample/condition tested. Data are presented as mean 6 SEM of three independent experiments. Differences between averages were analyzed by two-tailed Student's t test. Significance was set at p value of , 0.05. *p , 0.05; **p , 0.01; n.s. not significant. 1. Medzhitov, R. Janeway Jr, C. A. Innate immunity: the virtues of a nonclonal system of recognition. Cell 91, 295?98 (1997). 2. Palm, N. W. Medzhitov, R. Pattern recognition receptors and control of adaptive immunity. Immunological reviews 227, 221?33, doi:10.1111/j.1600065X.2008.00731.x (2009). 3. Malhotra, R. et al. Glycosylation changes of IgG associated with [https://www.medchemexpress.com/Y-27632-dihydrochloride.html Y-27632 In stock] rheumatoid arthritis can activate complement via the mannose-binding protein. Nature medicine 1, 237?43 (1995). 4. Arnold, J. N. et al. Human serum IgM glycosylation: identification of glycoforms that can bind to mannan-binding lectin. The Journal of biological chemistry 280, 29080?9087, doi:10.1074/jbc.M504528200 (2005). 5. Royle, L. et al. Secretory IgA N- and O-glycans provide a link between the innate and adaptive immune systems. J Biol Chem 278, 20140?0153, doi:10.1074/ jbc.M301436200 (2003). 6. Notkins, A. L. Polyreactivity of antibody molecules. Trends Immunol 25, 174?79, doi:10.1016/j.it.2004.02.004 (2004). 7. Zhou, Z. H. et al. The broad antibacterial activity of the natural antibody repertoire is due to polyreactive antibodies. Cell host microbe 1, 51?1, doi:10.1016/ j.chom.2007.01.002 (2007). 8. Ochsenbein, A. F. et al. Control of early viral and bacterial distribution and disease by natural antibodies. Science 286, 2156?159 (1999). 9. Ehrenstein, M. R. Notley, C. A. The importance of natural IgM: scavenger, protector and regulator. Nature reviews. Immunology 10, 778?86, doi:10.1038/ nri2849 (2010). 10. Boyden, S. V. Natural antibodies and the immune response. Advances in immunology 5, 1?8 (1966). 11. Michael, J. G. Natural antibodies. Current topics in microbiology and immunology 48, 43?2 (1969). 12. Panda, S., Zhang, J., Tan, N. S., Ho, B. | |
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รุ่นแก้ไขเมื่อ 22:37, 29 พฤศจิกายน 2564
Next, H-ficolin (200 nM) in running buffer was injected over the GlcNAc-immobilized chip for 100 s (association) followed by buffer flow for 200 s (dissociation). To characterize the binding of nIgG peptides to H-ficolin, separate second injections of increasing concentrations of nIgG peptides were made under similar running conditions. During the experiment, the flow rate was constantly maintained at 30 ml/min. The bound proteins were removed after one cycle by injecting 15 ml of 0.1 M NaOH (regeneration buffer). We used specific buffers for simulating the ``normal condition with TBS buffer (25 mM Tris, 145 mM NaCl, pH 7.4, 2.5 mM CaCl2) and the ``infection-inflammation condition with MBS buffer (25 mM MES, 145 mM NaCl, pH 6.5, 2 mM CaCl2). The SPR sensograms were analyzed by the BIAevaluation 3.2 software and the KD (dissociation constant) was calculated using 151 Langmuir binding model. The plots were finally made by overlaying the original binding curves (black) with the 151 binding model fitted curves (red). Controls used for normalization were obtained by injection of buffers alone instead of the proteins. The difference in the value of the resonance unit before and after injection is a measure of the peptide:protein interaction. The plots shown are representative of three independent experiments. Statistical analysis. For all the experiments, three replicates were performed per sample/condition tested. Data are presented as mean 6 SEM of three independent experiments. Differences between averages were analyzed by two-tailed Student's t test. Significance was set at p value of , 0.05. *p , 0.05; **p , 0.01; n.s. not significant. 1. Medzhitov, R. Janeway Jr, C. A. Innate immunity: the virtues of a nonclonal system of recognition. Cell 91, 295?98 (1997). 2. Palm, N. W. Medzhitov, R. Pattern recognition receptors and control of adaptive immunity. Immunological reviews 227, 221?33, doi:10.1111/j.1600065X.2008.00731.x (2009). 3. Malhotra, R. et al. Glycosylation changes of IgG associated with Y-27632 In stock rheumatoid arthritis can activate complement via the mannose-binding protein. Nature medicine 1, 237?43 (1995). 4. Arnold, J. N. et al. Human serum IgM glycosylation: identification of glycoforms that can bind to mannan-binding lectin. The Journal of biological chemistry 280, 29080?9087, doi:10.1074/jbc.M504528200 (2005). 5. Royle, L. et al. Secretory IgA N- and O-glycans provide a link between the innate and adaptive immune systems. J Biol Chem 278, 20140?0153, doi:10.1074/ jbc.M301436200 (2003). 6. Notkins, A. L. Polyreactivity of antibody molecules. Trends Immunol 25, 174?79, doi:10.1016/j.it.2004.02.004 (2004). 7. Zhou, Z. H. et al. The broad antibacterial activity of the natural antibody repertoire is due to polyreactive antibodies. Cell host microbe 1, 51?1, doi:10.1016/ j.chom.2007.01.002 (2007). 8. Ochsenbein, A. F. et al. Control of early viral and bacterial distribution and disease by natural antibodies. Science 286, 2156?159 (1999). 9. Ehrenstein, M. R. Notley, C. A. The importance of natural IgM: scavenger, protector and regulator. Nature reviews. Immunology 10, 778?86, doi:10.1038/ nri2849 (2010). 10. Boyden, S. V. Natural antibodies and the immune response. Advances in immunology 5, 1?8 (1966). 11. Michael, J. G. Natural antibodies. Current topics in microbiology and immunology 48, 43?2 (1969). 12. Panda, S., Zhang, J., Tan, N. S., Ho, B.
