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Anal Biochem. 1989 Oct;182(1):193-6.

Improving the quality of immunoblots by chromatography of polyclonal antisera on keratin affinity columns.

Author information

1
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.

Abstract

Unwanted reactivity of polyclonal antisera against keratins ("fingerprint proteins") is a problem commonly encountered when proteins transferred to nitrocellulose are studied by immunoblotting. Immunoreactivity against keratins is generally accompanied by a spotted background. This antikeratin immunoreactivity could be removed by adsorption of the antisera to human keratin bound to nitrocellulose. Larger amounts of antisera were purified from contaminant antikeratin antibodies by a single passage over a column of human keratin coupled to activated CH-Sepharose 4B. In contrast to nonpurified antisera and their IgG fractions, the column effluent no longer recognized the Mr 55,000-70,000 keratin proteins and exhibited a marked decrease in background labeling. We propose this simple method as a valuable alternative when affinity purification of polyclonal antisera on antigen columns is not practical.

PMID:
2481415
DOI:
10.1016/0003-2697(89)90741-0
[Indexed for MEDLINE]

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