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Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases
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JoVE Journal Biochimica
Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

DOI:
10.3791/64700-v

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09:07 min

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December 23, 2022

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, , , ,
1Department of Chemistry,Skidmore College

Capitoli

  • 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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Traduzione automatica

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.

Tags

Keywords: Concanavalin ASedimentation AssayGlucan PhosphatasesSubstrate BindingCarbohydrate-protein InteractionsSEX4 MutantsAmylopectinBinding BufferCentrifugationProtease Inhibitor

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