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Analysis of Escherichia coli ZinT by Differential Scanning Fluorimetry(DSF) (CAT#: STEM-MB-0831-WXH)

Introduction

Escherichia coli ZinT is a metal binding protein involved in zinc homeostasis, with additional putative functions in the resistance against other metals. ZinT is a 216-amino acid periplasmic protein expressed by Gram-negative bacteria, which shuttles Zn(II) ions to the ZnuABC transporter under zinc-limiting conditions.




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
85. Performing the scan

Materials

Real-time PCR instrument