Electrophoretic immunodesorption of proteins according to molecular weight by use of a double-membrane system
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A simple method is described for electrophoretic desorption of proteins from antigen-antibody complexes, with more than 90% recovery and without denaturation, after immunosorbent affinity chromatography. Radiolabeled or unlabeled human serum albumin (HSA) and α-1-antitrypsin (AAT), conjugated to rabbit anti-HSA or anti-AAT polyclonal antisera, respectively, were electrophoretically desorbed from Sepharose 4B. In addition, purification and concentration of the major HSA protein band (monomer) of 68 kD from the other oligomeric protein bands were achieved by use of a two-membrane system in a simple electroelution apparatus. The system consisted of an upper cellulose acetate membrane, with pore size 20 nm and separation limit 70 kD, and a lower dialysis cellophane membrane with molecular weight cut-off from 1–50 kD that cnables separation according to size. Furthermore, purification of the monomer HSA or AAT from normal human serum was performed with 92% recovery. Homogeneity was implied by the presence of one band after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, Western blot, and autoradiography.
Key WordsElectrophoretic immunodesorption Polyacrylamide gel electrophoresis Double-membrane system Human serum albumin Proteins
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- L. Hudson, F. Hay, Practical Immunology, Blackwell Scientific Publications, 3rd edn, 1989.Google Scholar
- A. L. Van Wezel, P. van der Marel, in J. C. Gribnau, J. Visser, R. J. F. Nivard, (Editors), Affinity Chromatography and Related Techniques, Elsevier Scientific, Amsterdam, 1982, p. 282.Google Scholar
- R. F. Atmeh, N. Abuharfeil, S. Sukhun, M. Abo-Shehada, Prep. Biochem.21, 1 (1991).Google Scholar
- O. H. Lowry, A. L. Rosebrough, A. L. Farr, R. J. Randall, J. Biol. Chem.139, 265 (1953).Google Scholar
- G. Furst, U. Stockerand, T. Denglr, Infusiontherapy24, 83 (1989).Google Scholar