Abstract
In this protocol, we used the T24 human bladder cancer cell line as a source of native antigens to construct fractionated lysate microarrays. Subsequently, these microarrays were used to compare the autoantibody responses of individuals with interstitial cystitis/painful bladder syndrome (IC/PBS) to those of normal female controls. To accomplish this, T24 cells were lysed under nondenaturing conditions to obtain native antigens. These native antigens were then fractionated in 2D using a PF-2D liquid chromatography; the first dimension separated the proteins by their isoelectric points, and the second separated them according to hydrophobicity. The resulting protein fractions were printed onto nitrocellulose-coated glass slides (PATH slides) to create a set of fractionated lysate microarrays. To compare the autoantibody responses of IC/PBS patients with normal controls, the fractionated lysate arrays were competitively hybridized with fluorescently labeled IgG samples purified from both IC/PBS and control sera. This protocol presents a detailed description of the creation and use of native antigen fractionated lysate microarrays for autoantibody profiling.
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Acknowledgments
This work was supported in part by grants DK063665, DK066020, DK075566 from the National Institutes of Health to B.C.S. Liu. Additional funding was supported by the Interstitial Cystitis Association and the Fishbein Family Foundation.
Disclosure: Timothy J. Barder, Ph.D. is the President and owner of Eprogen.
Bryce P. Nelson, Ph.D. is an employee and shareholder of Gentel Biosciences, Inc.
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Caiazzo, R.J., O’Rourke, D.J., Barder, T.J., Nelson, B.P., Liu, B.CS. (2011). Native Antigen Fractionation Protein Microarrays for Biomarker Discovery. In: Wu, C. (eds) Protein Microarray for Disease Analysis. Methods in Molecular Biology, vol 723. Humana Press. https://doi.org/10.1007/978-1-61779-043-0_9
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DOI: https://doi.org/10.1007/978-1-61779-043-0_9
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