Genetically engineering the N-terminus of alcohol dehydrogenase (ADH) onto a nor

Question

Genetically engineering the N-terminus of alcohol dehydrogenase (ADH) onto a normally cytosolic protein alters that protein's localization. The following western blot (detects proteins) was seen when cells were transfected with an ADH-actin chimeric construct and proteins were isolated from the cytosol and mitochondria. Antibod.es against actin (43 kDa), the cytosolic protein GAPDH (37 kDa), and the mitochondrial inner membrane protein succinate dehydrogenase A (72 kDa), were used in the analysis. Results of the western blot are shown below: How do you explain the presence of actin in two distinct subcellular pools of protein? Why is actin the same molecular mass in both pools? If the blot were stripped and reproved with an antibody against the N-terminal of alcohol dehydrogenase, where would you expect to find the signal?

Explanation / Answer

a. The presence of the signal in the cytosol is due to the cellular actin present in the cell and the signal from the mitochondria is due to the ADH-actin construct. As ADH is a mitochondrial enzyme.

b. The molecular mass being same in two pools can be due to the truncated expression of the ADH-actin construct in the cell. The molecular mass of actin in the cytosol is due to the cellular actin and molecular mass of the actin in the mitochondria can be due to the truncated version of the protein production in the cell.

c. If the blot is stripped and reprobed with anti-N-Ter ADH antibody, the protein band will be seen at 43Kda only as the molecular mass of this protein is due to the truncated protein from the construct.

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