Affinity purification of polyclonal antibodies from antigen immobilized in situ in sodium dodecyl sulfate-polyacrylamide gels.
A procedure for the preparation of affinity-purified antibody is described. Protein mixtures are separated by electrophoresis in sodium dodecyl sulfate (SDS)--polyacrylamide gels. Individual bands of protein are cut from the gel and fixed in situ with glutaraldehyde. The gel pieces are then homogenized and washed extensively with buffered solutions and chaotropic agents. The washed gels can then be used as immunoadsorbents to purify antibodies from crude antisera. This method should be especially useful for the preparation of small amounts of antibody to proteins that are difficult to purify by conventional means, that are available only in limited quantity, or that cannot be blotted to immunoadsorbents such as nitrocellulose or diazotized paper.
Madara
P J
PJ
Department of Animal Sciences, University of Maryland, College Park 20742.
Banghart
L R
LR
Jack
L J
LJ
Neira
L M
LM
Mather
I H
IH
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000918:Antibody Specificity; D000941:Antigens; D002462:Cell Membrane / Q000276:immunology; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005976:Glutaral; D006168:Guinea Pigs; D008321:Mammary Glands, Animal / Q000276:immunology; D008894:Milk Proteins / Q000276:immunology; D012967:Sodium Dodecyl Sulfate
D000906:Antibodies; D000941:Antigens; D003432:Cross-Linking Reagents; D008894:Milk Proteins; D012967:Sodium Dodecyl Sulfate; D005976:Glutaral