Question

3. Using a fluorescence microscope, you observed how the localization of GFP-Ras changes in response to the inhibitor.
   1. GFP is a reporter protein, what does this mean?

2. You used stains to visualize the nucleus and golgi bodies and the fluorescence microscope to determine whether the GFP-Ras protein was present in the golgi or nucleus. How do these stains work?
   1. Hoechst 34580

2. BODIPY TR Ceramide

Answer 1

Reporter protein: These are products of reporter genes used in gene expression studies. Expression of reporter protein is an indication of efficient transcriptional and translational activity of the desired gene. Reporter genes may be tagged to the gene of interest.Therefore when the gene of interest is expressed, reporter gene also expressed and thus provide an evidence of the expression of the gene of interest. GFP or Green Fluorescent Protein is a reporter protein. The gene for GFP protein is found in many marine organisms such as jelly fish Aequorea vctoria. GFP exhibits fluorescent green colour under blue-UV range of light

Hoechst 34580: It is a blue fluorescent dye. these dyes can bind to the nucleic adids of prokaryotes and eukaryotes. They bind to the minor grove of DNA double strand where Adenine and Thymine are rich. Hoechst dyes blue or cyan fluorescent colour under Ultra violet light. These dyes can be used to study the genomic DNA of live and dividing cells

BODIPY TR Ceramide: BODIPY (Boron dipyrromethene)TR Ceramide is red-fluorescent marker dye. It is commonly used in selective staining and labelling of Golgi apparatus in live cells. This stain help to understand lipid trafficking in cells. This fluorescent stain when excited at 568 nm, produce yellow/orange/red colour. This colour emission is studied using a fluorascent microscope