How you could use the antibody and cell fractionation

Reference no: EM133385786

There are four steps involved in studying a gene of interest. These steps are:

isolation of the gene of interest
insertion of the gene in a carrier piece of DNA (a vector) that enables its introduction into, and expression within, the target cells
introduction of the expression vector-plus-gene construct into cells
selection of cells expressing the gene of interest.

Consider that you are going to conduct a study of a gene to examine the cellular location and function of the protein encoded by the gene.

a. For step 1, you need to isolate, and amplify, the gene of interest so that you can put it into an expression vector. Assuming you knew the DNA sequence of the gene of interest, which technique could be used to amplify the gene? How would you check to ensure that you had amplified the gene of interest?

b. The expression vector that you are going to use in step 2 is a bacterial plasmid. Briefly explain what a plasmid is, and why the following components are necessary to make a complete expression vector:

i. a promoter
ii. an origin of replication
iii. your gene of interest
iv. a reporter protein or other marker protein.

c .In step 3, you intend to introduce your complete plasmid expression vector into a mammalian cell type that does not normally express the protein encoded by your gene of interest. Name, and briefly explain, the technique that is commonly used to allow plasmid expression vectors to be taken up by mammalian cells.

d. Step 3 is successful! The mammalian cells have taken up the plasmid expression vector and have started expressing the protein encoded by your gene of interest. You now want to find out where the protein is located inside the cells. Fortunately, you have an antibody to the protein encoded by your gene of interest. Briefly explain, in no more than 200 words, how you could use the antibody and cell fractionation techniques to establish where the protein is located within the cells.

Reference no: EM133385786

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