title: Protein purification-free method of binding affinity determination by microscale thermophoresis.
abstract: Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.
authors: Cancer and Inflammation Program, National Cancer Institute, USA.
authors: Lyuba
authors: L
authors: Khavrutskii
authors: Joanna
authors: J
authors: Yeh
authors: Olga
authors: O
authors: Timofeeva
authors: Sergey G
authors: SG
authors: Tarasov
authors: Samuel
authors: S
authors: Pritt
authors: Karen
authors: K
authors: Stefanisko
authors: Nadya
authors: N
authors: Tarasova
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D049452:Green Fluorescent Proteins
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