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An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
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Biochemistry
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JoVE Journal Biochemistry
An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

DOI:
10.3791/63665-v

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07:45 min

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June 06, 2022

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, , , , , , ,
1Department of Pathology,University of New Mexico School of Medicine, 2Department of Physics and Astronomy,University of New Mexico, 3Comprehensive Cancer Center,University of New Mexico Health Sciences Center

Chapters

  • 00:04Introduction
  • 00:53Coverslip Preparation
  • 03:18Functionalization of Array with Biotinylated Antibody
  • 04:09SiMPull of Proteins of Interest from Whole-Cell Lysates
  • 04:40Image Acquisition
  • 05:45Results: Capture of EGFR-GFP from Protein Lysates Using SiMPull
  • 07:19Conclusion

Summary

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The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Tags

AI-powered Protein Phosphorylation QuantificationSingle-molecule Pull-down AssaySiMPull ProtocolAntibody LabelingAutofluorescence ReductionSingle-molecule AnalysisPhosphorylation Status Of Individual ProteinsAlternative To Western Blotting And Mass Spectrometry

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