Analysis Interactions of the N-Terminal His-Tagged 6-Methyladenine (m6A) Reader Protein YTHDF2 with m6A Modified and Unmodified RNA by Microscale Thermophoresis (MST) (CAT#: STEM-MB-2461-LGZ)

Introduction

Microscale Thermophoresis (MST) is a technique for the quantitative analysis of interactions between biomolecules with low sample consumption. MST utilizes the local temperature field to measure the diffusion rates of the free and bound states of the fluorescently labeled protein, and determines the dissociation constant, KD, by fitting the binding isotherm with a 1:1 binding model. MST can be used to quantitative analysis of the interaction of the N-terminal his-tagged 6-methyladenine (m6A) reader protein YTHDF2 with m6A modified and unmodified RNA, in single-strand configuration or with RNA:DNA hybrid substrates.

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Principle Applications Procedure Materials Notes Related Products

Principle

One of the interacting molecules (mostly proteins) is labeled with fluorescent dye or combined with GFP label. The labeled protein and ligand molecules are placed in the capillary according to a specific concentration gradient. Infrared laser heating generates a microscopic temperature gradient field to undergo thermophoresis, with which the hydration layer, molecular size, electric charge and other molecular properties will change. Then the fluorescence distribution in the reaction system changes. In addition to accurately detecting interactions between biomolecules, MST can also obtain other parameters related to interactions between biomolecules by calculating dissociation constants (Ks), and achieve accurate qualitative analysis.

Applications

For characterizing thermodynamic parameters of biomolecular interactions.

Procedure

1. Sample processing.
2. MST detection.
3. Data analysis.

Materials

• Sample Type: protein 10uM/ 100ul or 50 micrograms, small molecules 100uM/200ul, protein peptides need to be sent at low temperature

Notes

In order to ensure the reliability of the test, some samples should be provided as much as possible.

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