You are studying a cytosolic protein; your friend is studying a nuclear localiza

Question

You are studying a cytosolic protein; your friend is studying a nuclear localization signal. How could you work together to show that his/her NLS is sufficient to cause any protein, including yours, to move to the nucleus. This will require several experiments (you must describe at least 3 experiments – think about showing what is specifically asked – think about control experiments). Please describe three experiments. So far, I have looked into protein localization using GFP and immunofluorescence but please expand on how these techniques could be used as an experiment and what a control experiment might look like for each experiment.

Explanation / Answer

Immunofluroscence - This technique is used to detect the exact location of the protein or the target molecule of interest. Here antibodies are used to bind to the protein which is coupled to a fluroscent probe. On staining the the immunofluroscence can be seen in confocal microscope. Here there are 2 methods. 1. Direct fluroscence 2. Indirect fluroscence. In direct fluroscence only the primary antibody is used which direct attaches to the protein of interest . But this method is less commonly used because the amount of primary antibody used is more and it is expensive. In indirect fluroscence method the secondary antibody is used which is coupled to primary antibody. Here the secondary antibody contains the fluroscent probe. This method allows the many secondary antibodies to attach itself to the primary antibody and hence the signal is strong. hence this method is preffered as it is less expensive. In GFP , which is green fluroscent protein obtained from jelly fish is used as a fluroscent probe. So , how you can detect that your protein when contains NLS sequence is targeted to the nucleus?? You have to attach NLS sequence to your protein along with fluroscent probe . On staining you can detect the fluroscence in the microscope. So how will you attach the NLS sequence ?? NLS sequence is usually contains many positive aminoacid residues like argine , valine , lysine . You have to detect your own NLS sequence using addition and deletion mutants to detect which region is needed for localisation and which is not required. So now you have to construct a plasmid with the NLS of your friend's protein and your cytosolic protein and a fluroscent marker like GFP. This recombinant protein now contains NLS and GFP probe which localises itself to the nucleus and you can detect it in the confocal microscope. Similarly you can use it for immunofluroscence also. For immunofluroscence you have to get primary antibody for your protein and also secondary antibody which attaches to the primary antibody and produce fluroscence. Your control experiment should be the same plasmid with no NLS sequence. The plasmid should only contain the cytosolic protein and fluroscent probe. Thus you can detect the protein travelled to nucleus with the help of NLS.

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