Improving the quality of immunoblots by chromatography of polyclonal antisera on keratin affinity columns.
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.
Girault
J A
JA
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.
Gorelick
F S
FS
Greengard
P
P
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D003101:Collodion; D006801:Humans; D007106:Immune Sera / Q000302:isolation & purification*; D015151:Immunoblotting / Q000592:standards; D007633:Keratins / Q000276:immunology; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
D000906:Antibodies; D007106:Immune Sera; D007633:Keratins; D003101:Collodion