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蛋白质纯化方法测定结合亲和力微尺度热泳

JoVE Journal
Biologia

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JoVE Journal Biologia

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

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蛋白质纯化方法测定结合亲和力微尺度热泳

DOI:

10.3791/50541-v

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10:22 min

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August 15, 2013

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Lyuba Khavrutskii², Joanna Yeh, Olga Timofeeva, Sergey G. Tarasov, Samuel Pritt, Karen Stefanisko, Nadya Tarasova

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

Tadução automática

可广泛用于微型热泳（MST）无需纯化从细胞裂解液中的目标蛋白的结合亲和力的测定。该协议涉及过表达GFP融合蛋白，在非变性条件下的细胞裂解，MST信号检测在不同浓度的配体的存在下。

Tags

Protein Purification Microscale Thermophoresis Binding Affinity KD Determination GFP-fused Protein STAT3 Cell Lysate Non-denaturing Conditions Drug Discovery Protein Interactions

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