Use of an SDS-gel-separated protein band as a ligand for affinity chromatography: procedure and application to the purification of domain-specific antibodies against alpha-actinin.
This paper describes a simple and efficient method for preparing affinity columns. We used protein separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis as a ligand. Protein bands detected in a polyacrylamide gel were electrophoretically transferred to CNBr-activated Sepharose using a buffer containing Nonidet P-40. The amount of ligand protein coupled to activated Sepharose by our method was almost comparable to that obtained by conventional coupling procedure with a native ligand protein. Using affinity columns prepared by this method, we have successfully purified anti-alpha-actinin antibody and antibodies highly specific to the rod domain of alpha-actinin from the antiserum. This new method should be useful for separating a specific antibody from an antiserum that has been raised against multiple antigens. In addition, the use of the SDS-gel-fractionated band facilitates the coupling of proteins that have low solubility under the coupling conditions.
Yoshihara
Y
Y
Department of Biology, Faculty of Sciences, Shimane University.
Kuroda
M
M
D000185:Actinin / Q000276:immunology* / Q000302:isolation & purification; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000918:Antibody Specificity; D002645:Chickens; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D003488:Cyanogen Bromide; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods; D008024:Ligands; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols; D012685:Sepharose; D012967:Sodium Dodecyl Sulfate
D000906:Antibodies; D003902:Detergents; D008024:Ligands; D000185:Actinin; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D017830:Octoxynol; D012685:Sepharose; C010615:Nonidet P-40; D003488:Cyanogen Bromide