Question

a) Fluorescent proteins such as green fluorescent protein (GFP) are commonly used in microscopy experiments. Describing the process of fluorescence, what is GFP and what is the main benefit of using fluorescent proteins over other fluorophores?

b) Describe how fluorescent proteins can be used to (i) study a protein’s gene expression and (ii) localise proteins of interest within cells?

c) The resolution of a standard confocal microscope is approximately 200nm. Describe an experiment that can be used to show whether two proteins are co-localised and/or interacting with one another without changing the microscope you are using?

Answer 1

a)The GFP, green fluorescent protein is a protein isolated firstly from the jellyfish Aequorea victoria which consists of 238 amino acid residues that exhibits bright green fluorescence when exposed to light. The light can be visualised by illuminating with blue light to UV rays. It is used in fluorescent microscopy to visualise the different cells or biomolecules of interest . It is used as a molecular marker in the field of cell and molecular biology.

Fluorescence is the property of a substance to emit the light that has absorbed some sort of light or electromagnetic radiation. GFP has various advantages over other fluorophores as it is inheritable, once it is introduced in an organism it will be passed to next generation and can help in study of gene transfer . it can diffuse readily through the cells. And most important it doesn't interfere with the normal biological processes.

b). i) fluorescent proteins can be used to study a protein’s gene expression by the following means:-

The fusion proteins containing GFP are introduced in the cell indiretly by using RNA of the construct for this mRNA encoding that protein is labelled or directly by tagging the protein itself. This method is useful for studying structural and functional characteristics of the tagged protein on a macromolecular or single-molecule scale with fluorescence microscopy

ii) localisation of proteins of interest within the cell-

the intrested proteins will be labelled with th GFP or the gene encoding for that particular protein will be tagged and introduced into the cell. Then the cells where the gene is expressed, and the tagged proteins are produced they will show the fluorescence and rest will not and we will be able to localise the particular protein of interest.

c)An experiment that can be used to show whether two proteins are co-localised and/or interacting with one another are as followed:-

split GFP process is utilised for this co-localisation of proteins. The protein is splittwd into two fragments which are able to self-assemble, and then each of these are fused to the two proteins of interest.These incomplete GFP fragments are unable to fluoresce alone. But, if the two proteins of interest get colocalized ,then the two GFP fragments assemble together to form a GFP-like structure which is able to fluoresce. Thus, by measuring the level of fluorescence it is possible to find whether the two proteins of interest colocalize interact or not.