Assume you find a gene that is upregulated in mice brains in response to marijua

Question

Assume you find a gene that is upregulated in mice brains in response to marijuana. How would you determine exactly where in the mouse brain this gene is expressed and to what extent? In addition, how would you determine the subcellular localization of the protein corresponding to this gene? Briefly, outline five experiments to address these questions. (15 points, 1 page maximum not including references; you will need to search the literature for biochemical techniques that would be appropriate for this.) 4. this gne is expressed and to wha to this gene? Briefly, outine

Explanation / Answer

To find exactky where in mouse brain this gene expressesd we can do a immuno histochemistry, this experiment is like, take differeent parts of the mouse barin with and without the treatment of marijuana,than probe a primery Antibody specific for that protein than probe a secondry Antibody which can give flurocence and specifically binds to the FC reagion of the primary Antibody. than we can visualize the protein by using flurocent microscopy.(confocal)

With the same experiment we can see, with what extent the protein is expressed in different parts of the barin by measuring the fluroscence. Onother way to check this is doing an RT-qPCR this can tell the ptrotein expression in quantitative manner but at m-RNA level not at protein level.(with or without the marijuina )

once we came know where this pretein is expressed specifically,than we can shift our experiment to the specific cell lines

.To check its subcellular localization we can do different experiments one is immunofluroscence,

for example we are assuming that our protein is a mitochondrial ptotein,so who to prove this?

Take a primary antibody specific to our proyein of intrest probe it.than probe a flurocently labeled (GREEN) secondry Antibody. Next select a mitochondrial resident and protein probe with a primary Antibody than probe with a flurocently labeled (RED) secondry Antibody, than visualize the cell by using confocal microscopy, if our protein of intrast is localizing to mitochondria we can see its colocalization with actual mitochondrial protein( RED)

We can see the co localization fof RED and GREEN colours.

We have to repeat the same experiment for all the subcellular organelles.

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