Abstract
Recent advances in the micropurification of polypeptides for structural analysis have centered upon individual improvements in the areas of HPLC (microbore), electrophoresis (electroblot) and bioaffinity chromatography (monoclonal antibody-immunoaffinity). With the advent of monoclonal antibody technology, immunoaffinity chromatography is rapidly becoming the most powerful single separation method for the complete, rapid purification of a protein of interest from complex mixtures of polypeptides. The numerous advantages of this method include its selectivity and the ability to efficiently recover polypeptides, which exist in trace concentrations, from crude cell lysates. Through the judicious selection of buffer and elution conditions, immunoaffinity techniques can often be linked with other micropurification methods such as SDS-PAGE and/or HPLC to eliminate intermediate handling steps and thus streamline overall purification schemes. This report will focus on pertinent aspects of optimization in the preparation and use of immunoaffinity columns for the microscale purification of polypeptides for microstructural analysis. Preferred methods for the selection and purification of monoclonal antibodies will be outlined in addition to an evaluation of immobilization methods. Several successful application of monoclonal immunoaffinity chromatography to the purification of polypeptides for microsequence analysis will be also be included.
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References
P. Cuatrecasas and C.B. Anfinsen (1971) in Meth. in Enzymology 22, 345–378.
G. Kohler and C. Milstein (1975) Nature 256, 495–497.
E. Engvall and P. Perlmann (1971) Immunochem. 8, 871–874.
S.W. Kessler (1975) J. Immunol. 115, 1617–1624.
W.N. Burnett (1981) Anal. Biochem. 112, 195–203.
J.J. LfItalien, in J. Shively (Editor), Methods of Protein Micro-Characterization, Humana Press, Clifton, N.J., 1986. Ch. 10, pp. 279–314.
C.C. Patrick and G. Virella (1978) Immunochem 15, 137–139.
M.R. Mackenzie, N.L. Warner, and G.F. Mitchell (1978) J. Immunol. 120, 1493–1496.
H. Juarez-Salinas, S. Engelhorn, W.L. Bigbee, M.A. Lowry and L.H. Stanker (1984) Bio Techniques 2, 164–169.
M.J. Gemski, B.P. Doctor, M.K. Gentry, M.G. Pluskal and M.P. Strickler (1985) Bio Techniques 3, 378–384.
Bio-Rad Bulletin 1085. Activated Affinity Supports:Affi-Gel 10 and 15 (1982) Bio-Rad Laboratories.
R.M. Hewick, M.W. Hunkapiller, L.E. Hood, and W.J. Dreyer, (1981), J. Biol. Chem. 256, 7990–7997.
J.J. L’Italien and R.A. Laursen (1981) J. Biol. Chem. 256, 8092–8101.
In Affinity Chromatography, Principles and Methods Pharmacia Ljungfortagan AB, Orebro, Sweden, 1983, pp 48–57.
U.K. Laemmli, (1970) Nature (London), 227, 680–685.
T.E. Zamb, (1985) in preparation.
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© 1987 Plenum Press, New York
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L’Italien, J.J. (1987). Application of Immunoaffinity Chromatography to the Purification of Polypeptides for Microsequence Analysis. In: L’Italien, J.J. (eds) Proteins. Springer, Boston, MA. https://doi.org/10.1007/978-1-4613-1787-6_10
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DOI: https://doi.org/10.1007/978-1-4613-1787-6_10
Publisher Name: Springer, Boston, MA
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