Particulate nitrocellulose as a solid phase for protein immobilization in immuno-affinity chromatography.
The use of nitrocellulose paper as a solid phase matrix for protein immobilization and its application in immunoaffinity chromatography is described. Pieces of nitrocellulose paper were frozen in liquid nitrogen and ground to a powder (NCP) which was then fractionated according to particle size by repeated sedimentation/resuspension cycles in water. The flow properties of different NCP fractions were compared with those of Sephadex G-50. Protein binding capacity and binding dynamics were investigated in a model system with bovine serum albumin (BSA) in phosphate buffered saline. Applications of the material are illustrated by batch chromatography for the removal of anti-carrier protein antibodies from a hapten antiserum and by affinity purification of a hapten-specific antibody fraction using NaSCN elution from haptenated NCP. Furthermore, the applicability of the material in column chromatography is demonstrated by elution of a monospecific fraction of anti-fluorescein antibodies from a hapten/carrier mixed bed column by excess of soluble hapten. The results demonstrate that NCP chromatography appears to be a cheap and useful alternative to many other chromatographic media used for protein immobilization.
Hammerl
P
P
University of Salzburg, Department of Biochemistry, Austria.
Hartl
A
A
Thalhamer
J
J
D000818:Animals; D000907:Antibodies, Bacterial / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods*; D003101:Collodion; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000276:immunology; D015151:Immunoblotting; D007136:Immunoglobulins / Q000302:isolation & purification; D008024:Ligands; D011485:Protein Binding; D011817:Rabbits
D000907:Antibodies, Bacterial; D007136:Immunoglobulins; D008024:Ligands; D003101:Collodion