AI Article Synopsis
- Developed a new method to measure protein binding affinity using microscale thermophoresis (MST) without needing purified proteins, making it quicker and less costly.
- The protocol involves overexpressing a GFP-fused protein and lysing cells under gentle conditions, allowing for easier testing of interactions.
- Successfully applied this method to determine the binding affinity of the transcription factor STAT3-GFP to various DNA sequences, which could open new avenues for studying diverse protein interactions.
Article 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.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3846881 | PMC |
| http://dx.doi.org/10.3791/50541 | DOI Listing |
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