Artifactual immunofluorescent labelling in yeast, demonstrated by affinity purification of antibody.
In the course of making antibodies against various yeast (S. cerevisiae) proteins, we have noted that it is common to observe reactivity of rabbit sera with a number of extraneous bands on Western transfers of yeast proteins. The pattern of reactive bands can change within a period of weeks, even when the rabbit has not been injected with antigen. A simple method of affinity purification, using antigen bound to nitrocellulose, is employed to remove the reactivity with these extraneous bands from immune sera. The importance of affinity purification is demonstrated by our attempts to immunolocalize a 55 kd yeast protein (p55). Immune serum stains yeast cells to give a striking pattern of spots and blotches not seen with preimmune serum. However, affinity purification of anti-p55 antibody shows that this pattern is not due to staining by anti-p55 antibody; rather the pattern is due to staining left in the serum depleted of anti-p55 antibody.
Lillie
S H
SH
Department of Anatomy and Cell Biology, University of Michigan Medical School, Ann Arbor 48109.
Brown
S S
SS
D000908:Antibodies, Fungal / Q000032:analysis / Q000302:isolation & purification*; D015153:Blotting, Western; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000276:immunology; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000032:analysis / Q000276:immunology*; D007106:Immune Sera / Q000032:analysis; D007074:Immunoglobulin G / Q000032:analysis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000032:analysis / Q000276:immunology*
D000908:Antibodies, Fungal; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; D007106:Immune Sera; D007074:Immunoglobulin G