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Analysis of Staphylococcus aureus FhuD2 by Differential Scanning Fluorimetry(DSF) (CAT#: STEM-MB-0830-WXH)

Introduction

Staphylococcus aureus is a human pathogen causing globally significant morbidity and mortality. The development of antibiotic resistance in S. aureus highlights the need for a preventive vaccine.<br />Iron availability plays an essential role in staphylococcal pathogenesis. We selected FhuD2, a lipoprotein involved in iron-hydroxamate uptake, as a novel vaccine candidate against Staphylococcus aureus. Unprecedented for staphylococcal lipoproteins, the protein was demonstrated to have a discrete, punctate localization on the bacterial surface. FhuD2 vaccination generated protective immunity against diverse clinical S. aureus isolates in murine infection models.




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
84. Performing the scan

Materials

Real-time PCR instrument