Identification of Polyol‐Responsive Monoclonal Antibodies for Use in Immunoaffinity Chromatography

Nancy E. Thompson1, Richard R. Burgess1

1 University of Wisconsin, Madison, Wisconsin
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 11.18
DOI:  10.1002/0471142727.mb1118s54
Online Posting Date:  May, 2001
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One of the limitations of immunoaffinity chromatography as been that high‐affinity antigen‐antibody complexes are difficult to dissociate, often leading to inactivation of the protein product during elution from the immobilized antibody. As described in this unit, some antigen‐antibody complexes can be dissociated in the presence of a combination of a low‐molecular‐weight polyhydroxylated compound (polyol) and a nonchaotropic salt. These conditions seem to be generally nondenaturing and, in some cases, even protein‐stabilizing. This type of antibody is designated “polyol‐responsive.“ These antibodies can be easily identified and isolated as monoclonal antibodies (MAbs) from a typical fusion, using standard hybridoma procedures. They have proven to be very valuable reagents for the immunoaffinity purification of active, labile, multi‐subunit protein complexes.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Purified or partially purified immunogen
  • Mice for immunization (unit 11.4)
  • recipe1× phosphate buffered saline (PBS; see recipe for 10×)
  • HAT medium (unit 11.6)
  • recipe1% nonfat dry milk (see recipe)
  • recipePBST (see recipe)
  • recipe1× TE buffer, pH 7.9 (see recipe for 10×)
  • recipe1× TE buffer, pH 7.9 (see recipe for 10×) containing salt‐polyol at appropriate concentration
  • Polyols (e.g., ethylene glycol, propylene glycol, 2, 3‐butanediol, glycerol)
  • Salts (e.g., ammonium sulfate, sodium chloride, potassium glutamate)
  • Anti‐mouse IgG conjugated to horseradish peroxidase (HRPO)
  • recipeOPD substrate (see recipe; or use other suitable substrate for HRPO)
  • 1 M H 2SO 4
  • Mouse sub‐isotyping kit (e.g., American Qualex; also see unit 11.3)
  • Protein A‐agarose (Repligen) or DE‐52 cellulose (Whatman)
  • Cyanogen bromide–activated Sepharose 4B (Sigma, or see unit 10.16)
  • 1 mM HCl
  • recipeBicarbonate coupling buffer (see recipe)
  • recipe1 M ethanolamine, pH 8.3 (see recipe)
  • recipeAcetate washing buffer (see recipe)
  • 2% (w/v) NaN 3
  • Crude material containing protein of interest
  • recipe1× TE buffer, pH 7.9 (see recipe for 10×) containing 2 M potassium thiocyanate (KSCN)
  • recipe1× TE buffer, pH 7.9 (see recipe for 10×) containing 100 mM NaCl and 0.02% NaN 3
  • 96‐well cell culture plates
  • Microtiter plate reader capable of reading at 490 nm
  • 30‐ml sintered glass filter and vacuum flask
  • End‐over‐end rotator
  • 10‐ml disposable polypropylene column (Bio‐Rad)
  • Additional reagents and equipment for production of monoclonal antibodies (units 11.4 11.11), ELISA (unit 11.2), immunoblotting (unit 10.8), preparation of antibody‐Sepharose (unit 10.16), dialysis ( 3.NaN), and protein assays (unit 10.1)
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Literature Cited

Literature Cited
   Cramer, P., Bushnell, D.A., Fu, J., Gnatt, A.L., Maier‐Davis, B., Thompson, N.E., Burgess, R.R., Edwards, A.M., David, P.R., and Kornberg, R.D. 2000. Architecture of RNA polymerase II and implications for the transcription mechanism. Science 288:640‐649.
   Edwards, A.M., Darst, S.A., Feaver, W.J., Thompson, N.E., Burgess, R.R., and Kornberg, R.D. 1990. Purification and lipid‐layer crystallization of yeast RNA polymerase II. Proc. Natl. Acad. Sci. U.S.A. 87:2122‐2126.
   Marshak, D.R., Kadonaga, J.T., Burgess, R.R., Knuth, M.W., Brennan, W.A. Jr., and Lin, S.‐H. 1996. Strategies for Protein Purification and Characterization. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Thompson, N.E. and Burgess, R.R. 1994. Purification of recombinant human transcription factor IIB by immunoaffinity chromatography. Protein Express. Purif. 5:468‐475.
   Thompson, N.E. and Burgess, R.R. 1999. Immunoaffinity purification of the RAP30 subunit of human transcription factor IIF. Protein Express. Purif. 17:260‐266.
   Thompson, N.E., Aronson, D.B., and Burgess, R.R. 1990. Purification of eukaryotic RNA polymerase II by immunoaffinity chromatography. J. Biol. Chem. 265:7069‐7077.
   Thompson, N.E., Hager, D.A., and Burgess, R.R. 1992. Isolation and characterization of a polyol‐responsive monoclonal antibody useful for gentle purification of Escherichia coli RNA polymerase. Biochemistry 31:7003‐7008.
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