b. Label which set of bands is the result of the amplifica- tion by the unc18 pr
ID: 187243 • Letter: B
Question
b. Label which set of bands is the result of the amplifica- tion by the unc18 primers and which by the GLUT4 primers. Why was RT-PCR with GLUT4 primers used as the positive control? What does this tell you about the relationship be- tween the knock down of the unc 18 and GLUT4 mRNAs?a c. With antibodies against unc18, draw a representative Western blot showing the expected results from protein sam-A ples from each of these five samples d. To further investigate the relationship between thedo unc18 and GLUT4 proteins, the siRNA experiment is re peated, but in embryonic cells expressing a GFP-tagged GLUT4 protein. Draw a cell including its mitochondria, nucleus, and rough endoplasmic reticulum and show where, if using a ties laser scanning confocal microscope, the GFP fluorescence would localize in the cells expressing the unc18 siRNAs. Do fun not forget to draw the cell representing the control. Bio e. The ability of a protein to colocalize with another pro- tein suggests but does not prove that the two physically inter- act with each other. Having each protein labeled, in this case Res unc18 to green fluorescent protein and GLUT4 to red fluores- cent protein, provides the experimenter with reagents to tease fluor apart the question of colocalization versus interaction. Using Biot these reagents, describe one technique that would simply denm onstrate unc18 colocalizes with GLUT4 and another tech-cenc nique that proves the two proteins interact physically and AOP more: References BioloExplanation / Answer
Ans. for the highlighted part.
Western blot is the technique to identify specific proteins from a complex mixture of proteins. The main three steps in this technique includes: separation by size, then transfer to a solid support, and marking target protein using a proper primary and secondary antibody to visualize.
It is visible that lower band from of the gel, has amplification in all the emryos, it means it is expressed in all the embyos, which suggests that it is GLUT4 as GLUT 4 is control and need to be amplified in all the cells.
While the upper band is of unc as it may or may not express in all the cells. By using antibodies against unc18 we will be able to block the western blotting with the specific antibody in order to prevent the nonspecific binding of this antibody to any other band or protein. Then after using the secondary antibody, we can visualize the protein on the membrane on which the gel bands has been transferred.
So we will get the exact copy of the upper band on the western blotting membrane as it is present on th SDS PAGE.
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