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Measuring Low-Picomolar Apparent Binding Affinities by Minigel Electrophoretic Mobility Shift.

Karen A Lewis1, Sarah E Altschuler2, Deborah S Wuttke3

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Methods in Molecular Biology (Clifton, N.J.)
|November 15, 2018
PubMed
Summary

Measuring high-affinity protein-DNA interactions, specifically telomere binding proteins, is challenging. This study details an electrophoretic mobility shift assay protocol for accurately measuring binding affinity constants in the low-picomolar range.

Keywords:
Binding assayDNA binding proteinEMSAHigh-affinity bindingNative gel

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • Accurate measurement of protein/DNA interactions with low-picomolar binding affinity constants is experimentally challenging.
  • Understanding telomere binding protein sequence specificity requires precise affinity measurements.

Purpose of the Study:

  • To develop and describe a robust electrophoretic mobility shift assay (EMSA) protocol.
  • To enable routine measurement of apparent dissociation constants (KD,app) in the 1-20 pM range for high-affinity protein-single-stranded DNA interactions.

Main Methods:

  • Adaptation of the electrophoretic mobility shift assay (EMSA).
  • Focus on specific experimental considerations for reproducible high-affinity measurements.
  • Utilizing the assay to probe sequence specificity of telomere binding proteins.

Main Results:

  • The developed EMSA protocol allows for routine measurement of KD,app values in the 1-20 pM range.
  • The protocol is effective for studying high-affinity interactions between proteins and single-stranded DNA.
  • Successful application in probing sequence specificity of telomere binding proteins.

Conclusions:

  • The described EMSA protocol overcomes the challenges of measuring low-picomolar binding affinities.
  • This method provides a reliable approach for characterizing high-affinity protein-DNA interactions.
  • The protocol is crucial for detailed studies of telomere binding protein function and specificity.