Summary
A method for the affinity purification of autoantibodies from small volumes of human sera using a western blot strip containing a target antigen electrophoretically transferred from a sodium dodecyl sulfate (SDS) polyacrylamide gel is described. This method is a very useful alternative for affinity column chromatography, particularly when the antigen of interest is of low abundance. The protein mixture is resolved on a preparative SDS polyacrylamide gel and transferred to nitrocellulose membrane. A couple of strips are excised vertically from either side of the blotted membrane and immunoblotted with specific antisera to identify the target band. Then the target band is excised horizontally and used for affinity purification. We have used this procedure to affinity purify antibodies to a 70,000 molecular weight protein derived from HeLa cell extract. A sham band, excised away from the target antigen, was used as a control for sham purification of autoantibodies. The autoantibodies purified in this manner reproduced the multiple nuclear dot anti-nuclear antibody pattern obtained using crude sera from 21 patients without primary biliary cirrhosis or anti-mitochondrial antibody.
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References
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Acknowledgment
This work was supported by NIH grant ARO1844 and Oklahoma Center for the Advancement of Science and Technology to Dr. Hal Scofield, OMRF, OKC, OK, USA.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Kurien, B.T. (2009). Affinity Purification of Autoantibodies from an Antigen Strip Excised from a Nitrocellulose Protein Blot. In: Kurien, B., Scofield, R. (eds) Protein Blotting and Detection. Methods in Molecular Biology, vol 536. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-542-8_22
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DOI: https://doi.org/10.1007/978-1-59745-542-8_22
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