Analysis of the diffusion of Ras2 in Saccharomyces cerevisiae by Fluorescence recovery after photobleaching (FRAP) (CAT#: STEM-MT-0039-WXH)

Name: Analysis of the diffusion of Ras2 in Saccharomyces cerevisiae by Fluorescence recovery after photobleaching (FRAP)
SKU: STEM-MT-0039-WXH
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Introduction

Ras2 is a Saccharomyces cerevisiae guanine nucleotide-binding protein (encoded by the RAS2 gene) which becomes activated by binding GTP when glucose is present in the environment. It affects growth regulation and starvation response.
RAS2 regulates spore formation via Ras/cyclic AMP/protein kinase A pathway. The expression of GPX1 is induced by glucose starvation and treatment with calcium chloride involving the Ras/cAMP pathway and Snf1.

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Principle Applications Procedure Materials

Principle

Fluorescence recovery after photobleaching (FRAP) is a microscopy technique capable of quantifying the mobility of molecules within cells. By exploiting the phenomenon of photobleaching, fluorescent mole- cules within a region of interest can be selectively and irreversibly 'turned off'. It is capable of quantifying the two-dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes, or to examine single cells.

Applications

• Characterization of the mobility of individual lipid molecules within a cell membrane.
• Analysis of molecule diffusion within the cell
• Study of protein interaction partners, organelle continuity and protein trafficking.

Procedure

1. An initial fluorescence of fluorescent molecules is measured in the region of interest (ROI).
2. The fluorescent molecules are rapidly photobleached by focusing the high-intensity laser beam onto the defined area.
3. The exchange of bleached molecules with unbleached molecules from the surrounding region is followed over time using a low-intensity laser.