Can I have someone provide me some feedbacj on this question for my cellular bio

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Can I have someone provide me some feedbacj on this question for my cellular biology course. Maybe give me some other information that you think I should include 4. 6pts. You hypothesize that a protein is localized to the mitochondria in a cell. Design a microscopy experiment to test this hypothesis. Please include the labels and types of microscopy you would use. A. Materials: To perform this experiment we will need the following reactants Two molecular tags will be constructed, one will be GFP and the other RFP. Each tag while be construct activated, since mamma insect viru The virus will be fused with the 5' region of pyruvate dehydrogenase's amino acid seque enzyme responsible for converting pyruvate to Acetly CoA where it will enter and interact with the mitochondria thus placing the tag. A specific wavelength will be used depending on the tag, if both GFP & RFP localize in the same location than that will indicate that the tag is in the mitochondria and has been placed correctly. A confocal microscope Live mammalian cells Agar growth medium incubated in an aerobic environment overnight at 37 degrees Celsius 1. ed via an insect virus to ensure that no antibody mediated immune response is lian cells are unable to recognize the viral glycoproteins located on the s's nuclear envelope, this will ensure that degradation of the two tags will not occur. 2. 3. 4. B. If you determine that the protein is NOT localized to the mitochondria, how might you find out if the protein localizes to another organelle. .Determine the molecular weight of each organelle via amino acid sequence, next we can use SDS-PAGE and obtain a band. Using both sets of data we can match up corresponding molecular weights and determine where that protein is located. C. How might you test this hypothesis without using a microscope technique? To test this hypothesis, we can use western blots. For this process, we will first use gel electrophoresis to separate and sort the various proteins in each of the organelles according to their physical characteristics. Next the separated protein bands will then be transferred to a nitrocellulose membrane and treated with antibodies that are specific for the protein in question, and other reactants. The primary antibody will bind to the protein of interest, and the secondary protein tag with the colorimetric signal will bind to the primary tag identifying the protein

Explanation / Answer

B.

Rate-zonal centrifugation or differential centrifugation or density gradient centrifugation can be used to separate the organelles.

Sequential fractionation and isolation of subcellular proteins from the mitochondria separated from the mammalian cells can be done. It is followed by the gel electrophoresis technique to identify the changed molecular weight of the protein carrying molecular tags.

C.

If the concerned protein is identified by gel electrophoresis, it can be separated properly and used for chemical analysis for its presence by supplying the substrate and see for its reaction. The molecular tags can be identified using western blot, flow cytometry, and imaging.

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