For homework, I had to prove that a certain catalyst was a protein, then tell how I would purify the protein, then tell how I would clone a human chromosomal region that contains the gene encoding the protein. The last part of the question is where I am stuck which says: how would you verify that one of the genes you isolated does indeed encode a protein with the catalytic properties of the protein being studied?
Can someone please give me a good, detailed explanation as to an experiment I could do to verify that the gene I cloned encodes the protein I am interested in?
Thank you!
Can someone please give me a good, detailed explanation as to an experiment I could do to verify that the gene I cloned encodes the protein I am interested in?
Thank you!
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How would you verify that one of the genes you isolated does indeed encode a protein with the catalytic properties of the protein being studied?
Do you know the function of the protein? If yes, you could silence the respective gene, thus resulting in no expression of the protein.Then, perform biochemical test.
For simplicity...
E.g. you integrate gene into vector, incorporated into host organism (bacteria), separate into two conditions, one of which you added repressor protein and the other, under normal conditions. Assuming that the expression system is perfect, the cells that are under normal conditions would produce the protein with catalytic properties. You then perform test on both samples.
Expected results:
[Control] Wild Type Cells: To assure that these cells does not have the gene X (encodes of protein X) initially.
[Control] Sample under normal conditions: Protein X + Substrate A = Product B
Sample with repressor protein (or anything else that silence the gene): With absence of gene expression, there should be no protein X produced, hence product B is not generated. This indicates that the gene isolated is associated with production of protein X
Hope this helps!
Do you know the function of the protein? If yes, you could silence the respective gene, thus resulting in no expression of the protein.Then, perform biochemical test.
For simplicity...
E.g. you integrate gene into vector, incorporated into host organism (bacteria), separate into two conditions, one of which you added repressor protein and the other, under normal conditions. Assuming that the expression system is perfect, the cells that are under normal conditions would produce the protein with catalytic properties. You then perform test on both samples.
Expected results:
[Control] Wild Type Cells: To assure that these cells does not have the gene X (encodes of protein X) initially.
[Control] Sample under normal conditions: Protein X + Substrate A = Product B
Sample with repressor protein (or anything else that silence the gene): With absence of gene expression, there should be no protein X produced, hence product B is not generated. This indicates that the gene isolated is associated with production of protein X
Hope this helps!