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Protein Purification-fri metode til Binding Affinity Bestemmelse ved Microscale Thermophoresis
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Biologia
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JoVE Journal Biologia
Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Protein Purification-fri metode til Binding Affinity Bestemmelse ved Microscale Thermophoresis

DOI:
10.3791/50541-v

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10:22 min

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August 15, 2013

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, , , , , ,
1Cancer and Inflammation Program,National Cancer Institute, 2Basic Science Program,SAIC-Frederick, Inc., 3Departments of Oncology and Radiation Medicine,Georgetown University Medical Center, 4Structural Biophysics Laboratory,National Cancer Institute

Capítulos

  • 00:05Título
  • 01:43Preparation of Cell Lysate
  • 03:20Determination of Optimal Lysate Dilution
  • 04:02Microscale Thermophoresis Binding Studies
  • 07:05Microscale Thermophoresis Data Analysis
  • 08:25Results: Measuring the Affinity of Non-phosphorylated STAT3 Protein Binding to Oligonucleotides
  • 09:40Conclusion

Summary

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Mikroskala thermophoresis (MST) kan anvendes bredt til bestemmelse af bindingsaffinitet uden oprensning af målproteinet fra cellelysater. Protokollen involverer overekspression af GFP-fusioneret protein, cellelyse i ikke-denaturerende betingelser, og påvisning af MST signal i nærvær af varierende koncentrationer af liganden.

Tags

Protein PurificationMicroscale ThermophoresisBinding AffinityKD DeterminationGFP-fused ProteinSTAT3Cell LysateNon-denaturing ConditionsDrug DiscoveryProtein Interactions

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