1. Consider the example of a study in which investigators examined the regulatio
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1. Consider the example of a study in which investigators examined the regulation of a drug-metabolizing gene called DDR. Inspection of the promoter region of DDR revealed consensus sequences for hepatocyte nuclear factor (HNF) proteins. Specifically, the researchers suspected that one member of this protein family, HNF-4 or HNF-1, was involved in gene regulation. For this, the research team decided to use electrophoretic mobility shift assay (EMSA) by making a radiolabeled DNA probe for the promoter region they were studying F: probe free DNA and NE: Nuclear Extract After combining the labeled probe with nuclear extract (denoted "N.E. in the figure below), the investigators noted a clear shift in the location of the probe (lane 2). Why does the shift in gel observed in the presence of nuclear extract? (5 pt) A. B. Next, the researchers performed a competition experiment using an excess of nlabeled DNA probe; in this case, the shift was no longer visible (lane 3). Why does this happen? (5 pt)Explanation / Answer
A. Electrophoretic mobility shift assay is the method that detect the detect protein complexes with nucleic acids. Due to interaction of protein with nucleic acid there is formation of complex structure larger in size that move slowly than the native DNA.
This is why after combining the radiolabelled probe with nucleic acid extract there is interaction between the probe and the nucleic acid that moves slower than the free nucleic acid extract.
B. While doing the competition experiment in which they use the excess of unlabeled DNA probe, there is no shift visible because the DNA will be visible under UV light in the gel after illumination. However the probe DNA interaction will be there, but due to unlabeled probe, it is not detected in the UV trans- illuminator like the DNA does. This is the reason that we can observe only native DNA not the NE + probe.
C. From the gel it can be seen that there is supershift in the lane 6. Yhis shows that there is the maximum interaction of the antibody to the DNA and the probe that is why it is seen topmost in the gel.
On the other hand there is no interaction of probe and DNA in the lane 4 as no supershift is visible.
Lane 7 has some level of binding of antibodies to the protein while some of the DNA remain binded to the protein but not to the antibody as there is slow movement than the Probe +DNA.
Similarly lane 5 and 8 has interaction of DNA with protein, but no specific binding of the antibody to the protein.
D. There is protein protein interaction visible in only lane 6. That is HNFalpha 4a with the nuclear extract and probe.
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