Determining the effect of urea by Differential Scanning Fluorimetry(DSF) (CAT#: STEM-MB-0785-WXH)

Introduction

Urea shifts the equilibrium from the native to denatured ensemble by making the protein-protein contact less stable than protein-urea contact, which is just the reverse of the condition in pure water, where protein-protein contact is more stable than protein-water contact. Urea also promoted protein unfolding in an indirect manner by altering water structure and dynamics, as also occurs on the introduction of nonpolar groups to water, thereby diminishing the hydrophobic effect and facilitating the exposure of the hydrophobic core residues.

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Principle Applications Procedure Materials Notes Related Products

Principle

Differential Scanning Fluorimetry measures protein thermal unfolding by monitoring changes in fluorescence emission of a sample upon heating. This allows the determination of protein thermostability and complex formation even with weakly binding ligands by thermal shift assay. Differential Scanning Fluorimetry is therefore ideally suited for screening of optimum buffer conditions like pH, buffer composition and ionic strength. The technique is applicable to any biological sample, from soluble proteins to integral membrane proteins.

Applications

To identify low-molecular-weight ligands that bind and stabilize purified proteins.
To measure the denaturation and unfolding of proteins.

Procedure

1. Preparation of compound solutions
2. Preparation of buffer/additive screen plates
3. Preparation of compound storage plates
4. Equipment preparation
5. Sample preparation
39. Performing the scan

Materials

Real-time PCR instrument

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