Immunoblotting of pollen proteins: Tween 20 mediates nonspecific binding of gammaglobulins to immunoblots of oak-pollen proteins.
Oak-pollen extracts, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose, demonstrated extensive banding in the absence of human sera or with nonatopic sera with both a horseradish peroxidase-conjugated goat antihuman IgE and 125I-labeled rabbit antihuman IgE. When the nonionic detergent Tween 20 was removed from all incubations and washes, negative banding appeared, corresponding to bands that were clearly visible when Tween 20 had been used as a blocking agent. When Tween 20 was reintroduced, the bands reappeared. These results demonstrate that, although Tween 20 is effective in blocking unbound sites on nitrocellulose membranes, this and other nonionic detergents may cause nonspecific binding to immunoblots of oak-pollen proteins. Thus, care must be used in the interpretation of immunoblots to detect allergens when Tween 20 is used as the blocking agent.
Loria
R C
RC
Washington University School of Medicine, Department of Medicine, St. Louis, MO 63110.
Prange
J O
JO
Wedner
H J
HJ
D000818:Animals; D001666:Binding Sites, Antibody / Q000187:drug effects; D001667:Binding, Competitive; D002364:Caseins; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D006041:Goats; D006735:Horseradish Peroxidase; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007073:Immunoglobulin E / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis*; D011058:Pollen / Q000032:analysis*; D011136:Polysorbates; D011817:Rabbits; D012521:Sarcosine / Q000031:analogs & derivatives; D013849:Thimerosal; D014197:Trees / Q000276:immunology*
D002364:Caseins; D010940:Plant Proteins; D011136:Polysorbates; D013849:Thimerosal; D007073:Immunoglobulin E; C025231:sarkosyl; D003101:Collodion; D006735:Horseradish Peroxidase; D012521:Sarcosine