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Related Experiment Videos

Proteolytic digestion of mouse IgE.

S Haba1, A Nisonoff

  • 1Department of Biology, Brandeis University, Waltham, MA 02254.

Journal of Immunological Methods
|April 8, 1991
PubMed
Summary

This study details methods for preparing mouse immunoglobulin E (IgE) fragments, F(ab’)2 and Fab. Pepsin and trypsin are recommended for F(ab’)2 preparation, while papain yields the best purified Fab fragments.

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Area of Science:

  • Immunology
  • Biochemistry
  • Protein Chemistry

Background:

  • Mouse immunoglobulin E (IgE) plays a crucial role in allergic responses.
  • The enzymatic digestion of IgE into fragments like F(ab’)2 and Fab is essential for various immunological applications.
  • Understanding the optimal conditions for generating these fragments is key for research and diagnostics.

Purpose of the Study:

  • To describe conditions for preparing F(ab’)2 and Fab fragments from mouse IgE.
  • To compare the efficacy of different proteases (papain, pepsin, trypsin) in fragment generation.
  • To identify the most suitable methods for obtaining high yields of purified fragments.

Main Methods:

  • Enzymatic digestion of mouse IgE using papain, pepsin, or trypsin.
  • Analysis of fragment sizes (Mr approximately 130,000 for F(ab’)2) using gel electrophoresis.
  • Further digestion of F(ab’)2 to yield Fab fragments.
  • Purification of Fab fragments.

Main Results:

  • All three enzymes produced F(ab’)2 fragments (Mr ~130,000) that could be further digested into Fab fragments.
  • Pepsin and trypsin were more suitable for F(ab’)2 preparation due to easier separation from by-products compared to papain.
  • Papain yielded the best results for purified Fab fragment preparation.
  • Digestion rates varied, with pepsin and trypsin being significantly faster than papain.

Conclusions:

  • Enzymatic digestion offers a viable method for producing mouse IgE F(ab’)2 and Fab fragments.
  • The choice of enzyme impacts the efficiency of F(ab’)2 preparation and Fab purification.
  • Pepsin and trypsin are preferred for F(ab’)2, while papain is optimal for Fab fragment yield.

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