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Analysis of RNA Helicases by Differential Scanning Fluorimetry(DSF) (CAT#: STEM-MB-0808-WXH)

Introduction

RNA helicases are essential for most processes of RNA metabolism such as ribosome biogenesis, pre-mRNA splicing, and translation initiation. They also play an important role in sensing viral RNAs. RNA helicases are involved in the mediation of antiviral immune response because they can identify foreign RNAs in vertebrates. About 80% of all viruses are RNA viruses and they contain their own RNA helicases. Defective RNA helicases have been linked to cancers, infectious diseases and neuro-degenerative disorders. Some neurological disorders associated with defective RNA helicases are: amyotrophic lateral sclerosis, spinal muscular atrophy, spinocerebellar ataxia type-2, Alzheimer disease, and lethal congenital contracture syndrome.




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
62. Performing the scan

Materials

Real-time PCR instrument
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