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

Anal Biochem. 1989 Oct;182(1):193-6. doi: 10.1016/0003-2697(89)90741-0.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies / isolation & purification*
  • Chromatography, Affinity / methods
  • Collodion
  • Humans
  • Immune Sera / isolation & purification*
  • Immunoblotting / standards
  • Keratins* / immunology
  • Male
  • Rats
  • Rats, Inbred Strains

Substances

  • Antibodies
  • Immune Sera
  • Keratins
  • Collodion