Affinity Precipitation Methods

  • Jane A. Irwin
  • Keith F. Tipton
Part of the Methods in Molecular Biology™ book series (MIMB, volume 59)


Affinity chromatography (see  Chapter 16) is a powerful protein purification technique, that exploits the specific interaction between a biological ligand (e.g., a substrate, coenzyme, hormone, antibody, or nucleic acid) or its synthetic analog and its complementary binding site on a protein. One of the variations on this technique (see refs. 1, 2, 3 for reviews) was that of affinity precipitation. As in affinity chromatography, the protein binds to a specific ligand, but the latter is free in solution, rather than bound to an insoluble support. Ligand binding gives rise to the precipitation of the protein from solution, which is then followed by centrifugation. The pellet contains the protein of interest and the ligand, whereas the other components of the mixture remain in the supernatant, allowing easy separation.


Potassium Phosphate Buffer Substrate Analog Phenazine Methosulfate HPLC Retention Time Pimelic Acid 
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  1. 1.
    Chen, J.-P. (1990) Novel affinity based processes for protein purification. J. Ferment. Bioeng. 70, 199–209.CrossRefGoogle Scholar
  2. 2.
    Luong, J. H. T. and Nguyen, A.-L. (1992) Novel separations based on affinity interactions. Adv. Biochem. Eng. Biotechnol. 47, 138–158.Google Scholar
  3. 3.
    Irwin, J. A and Tipton, K. F. (1995) Affinity precipitation: a novel approach to protein purification. Essays Biochem. 29, 137–156.PubMedGoogle Scholar
  4. 4.
    Larsson, P.-O. and Mosbach, K. (1979) Affinity precipitation of enzymes. FEBS Lett. 98, 333–338.PubMedCrossRefGoogle Scholar
  5. 5.
    Flygare, S., Griffin, T., Larsson, P.-O., and Mosbach, K. (1983) Affinity precipitation of dehydrogenases. Anal. Biochem. 133,409–416.PubMedCrossRefGoogle Scholar
  6. 6.
    Larsson, P.-O., Flygare, S., and Mosbach, K. (1984) Affinity precipitation of dehydrogenases. Methods Enzymol. 104, 364–369.PubMedCrossRefGoogle Scholar
  7. 7.
    Beattie, R. E., Graham, L. D., Griffin, T. O., and Tipton, K. F. (1985) Purification of NAD+-dependent dehydrogenases by affinity precipitation with adipo-N 2,N 2′-dihydrazido bis-(N 6-carboxymethyl-NAD+) (bis-NAD+). Biochem. Soc. Trans. 12, 433.Google Scholar
  8. 8.
    Buchanan, M., O’Dea, C. D., Griffin, T. O., and Tipton, K. F. (1989) Reversible cross-linking of alcohol and lactate dehydrogenases with the bifunctional reagent N 2, N 2′-adipodihydrazido-bis-(N 6-carboxymethyl-NAD+). Biochem. Soc Trans. 17, 422.Google Scholar
  9. 9.
    Graham, L. D., Griffin, T. O., Beatty, R. E., Mc Carthy, A. D., and Tipton, K. F. (1985) Purification of liver GDH by affinity precipitation and studies on its denaturation. Biochim. Biophys. Acta. 828, 266–269.PubMedCrossRefGoogle Scholar
  10. 10.
    Beattie, R. E., Buchanan, M., and Tipton, K. F. (1987) The synthesis of N 2,N 2′-adipodihydrazido-bis-(N 6-carboxymethyl-ATP) and its use in the purification of phosphofructokinase. Biochem. Soc. Trans. 15, 1043,1044.Google Scholar
  11. 11.
    Hayet, M. and Vijayalakshmi, M. A. (1986) Affinity precipitation of proteins using bis-dyes. J. Chromatogr. 376, 157–161.PubMedCrossRefGoogle Scholar
  12. 12.
    Lowe, C. R. and Pearson, J. C. (1983) Bio-mimetic dyes, in Affinity Chromatography and Biological Recognition (Chaiken, I. M., Wilchek, M., and Parikh, I., eds.), Academic, London, pp. 421–432.Google Scholar
  13. 13.
    Pearson, J. C., Burton, S. J., and Lowe, C. R. (1986) Affinity precipitation of lactate dehydrogenase with a triazine dye derivative: selective precipitation of rabbit muscle lactate dehydrogenase with a Procion Blue H-B analog. Anal. Biochem. 158, 382–389.PubMedCrossRefGoogle Scholar
  14. 14.
    Pearson, J. C., Clonis, Y. D., and Lowe, C. R. (1989) Preparative affinity preparation of L-lactate dehydrogenase. J. Biotechnol 11, 267–274.CrossRefGoogle Scholar
  15. 15.
    Lilius, G., Persson, M., Bulow, L., and Mosbach, K. (1991) Metal affinity precipitation of proteins carrying genetically attached polyhistidine affinity tails. Eur. J. Biochem. 198, 499–504.PubMedCrossRefGoogle Scholar
  16. 16.
    Van Dam, M. E., Wuenschell, G. E., and Arnold, F. H. (1989) Metal affinity precipitation of proteins. Biotechnol. Appl. Biochem. 11, 492–502.PubMedGoogle Scholar
  17. 17.
    Schneider, M., Guillot, C., and Lamy, B. (1981) The affinity preciprtation technique Application to the isolation and purification of trypsin from bovine pancreas. Ann. N. Y. Acad. Sci. 369, 257–263.PubMedCrossRefGoogle Scholar
  18. 18.
    Senstad, C. and Mattiasson, B. (1989) Affinity-precipitation using chitosan as ligand carrier. Biotechnol. Bioeng. 33, 216–220.PubMedCrossRefGoogle Scholar
  19. 19.
    Senstad, C. and Mattiasson, B. (1989) Purificatton of wheat germ agglutinin using affinity flocculation with chitosan and a subsequent centrifugation or flotation step. Biotechnol. Bioeng. 34, 387–393.PubMedCrossRefGoogle Scholar
  20. 20.
    Taniguchi, M., Kobayashi, M., Natsui, K., and Fujii, M. (1989) Purification of staphylococcal protein A by affinity precipitation using a reversibly soluble-insoluble polymer with human IgG as a ligand. J. Ferment. Bioeng. 68, 32–36.CrossRefGoogle Scholar
  21. 21.
    Kamihira, M., Kaul, R., and Mattiasson, B. (1992) Purification of recombinant protein A by aqueous two-phase extraction integrated with affinity precipitation. Biotechnol. Bioeng. 40, 1381–1387.PubMedCrossRefGoogle Scholar
  22. 22.
    Bradshaw, A. P. and Sturgeon, R. J. (1990) The synthesis of soluble polymer-ligand complexes for affinity precipitation studies. Biotechnol. Techniques. 4, 67–71.CrossRefGoogle Scholar
  23. 23.
    Senstad, C. and Mattiasson, B. (1989) Preparation of soluble affinity complexes by a second affinity interaction: a model study. Biotechnol. Appl. Biochem. 11, 41–48.PubMedGoogle Scholar
  24. 24.
    Linné, E., Garg, N., Kaul, R., and Mattiasson, B. (1992) Evaluation of alginate as a ligand carrier in affinity precipitation. Biotechnol. Appl. Biochem. 16, 48–56.Google Scholar
  25. 25.
    Gupta, M. N., Dong, G. Q., and Matiasson, B. (1993) purification of endopolygalacturonase by affinity precipitation using alginate. Biotechnol. Appl. Biochem. 18, 321–328.PubMedGoogle Scholar
  26. 26.
    Chen, J. P. and Hoffman, A. S. (1990) Polymer-protein conjugates II Affinity precipitation separation of human immunogammaglobulin by a poly (N-isopropylacrylamide)-protein A conjugate. Biomaterials. 11, 631–634.PubMedCrossRefGoogle Scholar
  27. 27.
    Nguyen, A. L. and Luong, J. H. T. (1989) Syntheses and applications of water-soluble reactive polymers for purification and immobilization of biomolecules. Biotechnol. Bioeng. 34, 1186–1190.PubMedCrossRefGoogle Scholar
  28. 28.
    Irwin, J. A. and Tipton, K. F. (1995) Resolution of lactate dehydrogenase isoforms by affinity precipitation. Biochem. Soc. Trans. 23, 3655.Google Scholar
  29. 29.
    Larsson, P.-O. and Mosbach, K. (1981) Novel affinity techniques. Biochem. Soc. Trans. 9, 285–287.Google Scholar
  30. 30.
    Feinstein, A. and Rowe, A. J. (1965) Molecular mechanism of formation of an antigen-antibody complex. Nature 205, 1473–149.CrossRefGoogle Scholar
  31. 31.
    O’Carra, P. (1978) Theory and practice of affinity chromatography, in Chromatography of Synthetic and Biological Polymers, 2nd ed. (Epton, R., ed.), Ellis Horwood for the Chemical Society, London, Chapter 11, pp. 131–158.Google Scholar
  32. 32.
    Bergmeyer, H. U., Grassl, M., and Walter, H.-E. (1983) Reagents for enzymatic analysis, in Methods of Enzymatic Analysis, vol 2, 3rd ed. (Bergmeyer, H. U., Bergmeyer, J., and Grassl, M., eds. ), Verlag Chemte, Wemheim, pp. 126–328.Google Scholar
  33. 33.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680–685.PubMedCrossRefGoogle Scholar
  34. 34.
    Mosbach, K., Larsson, P.-O., and Lowe, C. (1976) Immobilised coenzymes. Methods Enzymol. 44, 859–887.PubMedCrossRefGoogle Scholar
  35. 35.
    Engel, J. D. (1975) Mechanism of the Dimroth rearrangement in adenine. Biochem. Biophys. Res. Comm. 64, 581–585.PubMedCrossRefGoogle Scholar
  36. 36.
    Buchanan, M. (1988) The synthesis of N 2,N 2′-Adipodihydrazido-Bis-(N 6-Carboxymethyl-NAD)and N 2,N 2′-Adipodihydrazido-Bis-(N 6-Carboxymethyl-ATP) and Subsequent Affinity Precipitation of Enzymes. MSc Thesis, University of Dublin.Google Scholar
  37. 37.
    Beattie, R. E. (1984) The Synthesis of N 2,N 2′-Adipodihydrazido-Bis-(N 6-Carboxymethyl-NAD+) and its use in the Purification of Dehydrogenases. MSc Thesis, University of Dublin.Google Scholar
  38. 38.
    Buckmann, A. F. (1987) A new synthesis of coenzymically active water-soluble macromolecular NAD and NADP derivatives. Biocatalysis. 1, 173–186.CrossRefGoogle Scholar
  39. 39.
    Buckmann, A. F. and Wray, V. (1992) A simplified procedure for the synthesis and purification of N 6-(2-aminoethyl)-NAD and tricyclic 1, N 6-ethanoadenine NAD. Biotechnol. Appl. Biochem. 15, 303–310.Google Scholar
  40. 40.
    Butler, P. J. G. and Thelwall Jones, G. M. (1970) The preparation of alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase from baker’s yeast. Biochem. J. 118, 375–378.PubMedGoogle Scholar
  41. 41.
    Mc Carthy, A. D., Walker, J. M., and Tipton, K. F. (1980) Purification of glutamate dehydrogenase from ox brain and liver. Biochem. J. 191, 605–611.Google Scholar
  42. 42.
    Phelps, C. (1984) in Techniques in the Life Sciences: vol. B1/1, suppl. BS 104, Protein and Enzyme Biochemistry (Tipton, K. F., ed.), Elsevier, Shannon Industrial Estate, Ireland, pp. 1–16.Google Scholar

Copyright information

© Humana Press Inc. 1996

Authors and Affiliations

  • Jane A. Irwin
    • 1
  • Keith F. Tipton
    • 1
  1. 1.Department of BiochemistryTrinity CollegeDublinIreland

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