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Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases
JoVE 杂志
生物化学
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JoVE 杂志 生物化学
Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases
DOI:

09:07 min

December 23, 2022

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Chapters

  • 00:04Introduction
  • 01:03Preparation of ConA‐Sepharose Beads and ConA‐Sepharose: Amylopectin Beads
  • 02:19Starch Excess4 (SEX4) Protein Binding with ConA‐Sepharose: Amylopectin Beads
  • 03:41Running SDS‐PAGE Gels
  • 04:27Western Blotting for Chemiluminescence Detection and Data Analysis
  • 06:28Results: Binding Affinity of SEX4 to ConA‐Sepharose‐Amylopectin Beads
  • 08:18Conclusion

Summary

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This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.

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