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In class, during our work on Methods in Cell Biology, we int…

In class, during our work on Methods in Cell Biology, we introduced a protein called Mena that is involved in organizing the actin cytoskeleton. The examples shown in class related to Mena’s role in cell movement, by organizing actin at a cell’s leading edge. Many proteins have multiple functions within cells. Here you will analyze some experiments designed to determine whether Mena’s organization of the actin cytoskeleton is involved in proper formation of the structure of the Golgi apparatus. The data in this question are simplified from Tang et al, MBoC (2016).   The researchers explored a possible relationship between Mena and a protein known to regulate Golgi apparatus structure – GRASP65. To prepare for their experiments, the researchers made cells with a GFP protein included; in these cells GFP is not fused to any other protein (this is sample 1 in the experiment below). They made a separate set of cells with a Mena-GFP fusion protein included (this is sample 2 in the experiment below).   Next the researchers performed a co-immunoprecipitation experiment. They used antibodies that bind to GFP for the immunoprecipitation step, and they used antibodies that bind to GRASP65 for the western blot step.   a) (4 points) What protein/s does the band in lane 2 represent? What conclusions can you make from this lane? Explain briefly  b) (2 points) Why is there not a band present in the lane that contains sample 1? Explain briefly    Next, the researchers used a technique called siRNAs that can stop gene expression from a particular gene. (You do not need to know the details of how this technique works, just that the experimenters are using it to turn off gene expression from certain genes.) With this technique, they stopped the gene expression from the GRASP65 gene. They performed an immunofluorescence microscopy experiment using control cells that express GRASP65 (top row of images) and experimental cells in which expression from the gene encoding GRASP65 had been turned off (bottom row of images). They used antibodies that bind to Mena (shown in purple on the left), antibodies that bind to GRASP65 (shown in green, second from the left) and antibodies to a fluorescent dye that stains the Golgi apparatus (shown in red, second from the right). In each row all the images show the same cell, using different wavelengths of light for the different fluorescent dyes. c) (4 points) What can the researchers conclude from the top row of images – the wild-type cell? Explain briefly  d) (4 points) What can the researchers conclude from the bottom row of images – the cells in which gene expression from the GRASP65 gene has been stopped? Explain briefly  Make sure to answer all parts of the question and label the parts for the grader to identify each individually.  

You cut a plasmid with a restriction enzyme that cuts it int…

You cut a plasmid with a restriction enzyme that cuts it into three pieces — one fragment is 240 base pairs, one fragment is 600 base pairs, and one fragment is 1200 base pairs.  You carry out electrophoresis of the digested sample.  Which of the fragments will be closest to the top of the gel where the sample was loaded?