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Nitrocellulose-bound antigen repeatedly used for the affinity purification of specific polyclonal antibodies for screening DNA expression libraries.
- Source :
-
Journal of immunological methods [J Immunol Methods] 1988 Apr 06; Vol. 108 (1-2), pp. 115-22. - Publication Year :
- 1988
-
Abstract
- We present a simple, efficient and rapid method for affinity-purifying antibodies from a relatively crude antiserum in quantities large enough to screen a DNA expression library. The method presents a very convenient way to remove crossreacting or contaminating antibody specificities. The affinity matrix, antigen non-covalently bound to nitrocellulose, is prepared by the electrophoretic separation of antigen by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by the transfer of antigen to nitrocellulose. The matrix can be used repeatedly. A brief wash with 6 M guanidine hydrochloride is included between steps to remove residual antibodies which bind with high affinity to nitrocellulose-bound antigen. Various buffer solutions were assessed as antibody/antigen-dissociating agents. Glycine/HCl buffer, pH 2.5, appeared to be the most efficient in our hands, although a number of other less efficient dissociating reagents, including 4.5 M magnesium chloride, pH 7.5, 6 M urea, pH 7, and 0.05 M diethylamine, pH 11.5, also could be used; these may be the elution conditions of choice for other antibody/antigen combinations. The use of affinity-purified antibody solutions instead of the corresponding antisera gave increased signal-to-noise ratios with the detection systems that are commonly used to identify positive signals in screening expression libraries. Protein A- and goat anti-rabbit-alkaline phosphatase conjugates gave the most sensitive signals.
- Subjects :
- Animals
Brain Chemistry
Carbohydrate Epimerases genetics
DNA isolation & purification
Immune Sera isolation & purification
Intermediate Filament Proteins genetics
Neurofilament Proteins
Paper
Rats
Aldose-Ketose Isomerases
Antibodies isolation & purification
Antigens
Cloning, Molecular methods
Collodion
Electrophoresis, Polyacrylamide Gel methods
Immunoassay methods
Subjects
Details
- Language :
- English
- ISSN :
- 0022-1759
- Volume :
- 108
- Issue :
- 1-2
- Database :
- MEDLINE
- Journal :
- Journal of immunological methods
- Publication Type :
- Academic Journal
- Accession number :
- 3127468
- Full Text :
- https://doi.org/10.1016/0022-1759(88)90409-7