Immobilization of Proteins on Thionyl Chloride-Activated Controlled-Pore Glass

  • Violeta G. Janolino
  • Harold E. Swaisgood
Part of the Methods in Biotechnology book series (MIBT, volume 1)


Procedures for covalent immobilization should be capable of reacting with a limited number of surface residues of the protein under very mild conditions, preferably those that are optimal for the stability of the protein being immobilized. Sometimes it is also desirable to use a method of covalent immobilization that is reversible under mild conditions. We have developed the thionyl chloride-activated succinamidopropyl-glass as a matrix that provides these desirable properties (1, 2, 3). The activated support will react with only primary amino groups or sulfhydryl groups on the surface of the protein in physiological buffers in the pH range of 4.0–8.0 and in the cold room or at room temperature. Although the exact chemistry of the reaction still eludes definition, the required conditions and the reacting groups are well established. The thionyl chloride-activated succinamidopropyl surface can be stored for long periods of time (perhaps more than one year), and protein can be immobilized simply by incubating it with the matrix under the conditions chosen. The immobilized protein remains covalently bound even in the presence of strong denaturants, such as 6M guanidinium chloride; however, if desired, it can be partially removed by reaction with dilute solutions of hydroxylamine at room temperature (2). If the reaction was through amino groups, up to 80% of the protein can be removed. We have used this procedure both as a reversible method of covalent immobilization (2,4) and as a convenient as well as effective means for preparation of affinity matrices (5,6).


Glutamate Dehydrogenase Thionyl Chloride Silane Solution Succinic Anhydride Immobilize Protein 
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Copyright information

© Humana Press Inc , Totowa, NJ 1997

Authors and Affiliations

  • Violeta G. Janolino
    • 1
  • Harold E. Swaisgood
    • 1
  1. 1.Department of Food Science, College of Agriculture and Life SciencesNorth Carolinia State UniversityRaleigh

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