5.Genetically engineering the N?terminus of alcohol dehydrogenase (ADH) onto a n

Question

5.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. Antibodies 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 to the right:

a) How do you explain the presence of actin in two distinct subcellular pools of protein?

b) Why is actin the same molecular mass in both pools?

c) If the blot were stripped and re?probed with an antibody against the N?terminal of alcohol dehydrogenase, where would you expect to find the signal?

Explanation / Answer

a) Actin is present in mitochondria as well as cytosol. It has same molecular mass in both the pools. Actin combines with ADH to form a chimeric protein. This chimeric protein is functional in both the pools. Therefore, it is found in both the pools.

Please note that western blot is showing the three antibodies and not ADH-actin complex.

b) Actin is same. It has not changed. There are no two isoforms of Actin. So, it is same in both the pools.

c) Antibody against the N-terminal of alcohol dehydrogenase is applied. The signal would be found at both the 43kDa band.

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