706_F06pset1_ans - 7.06 FALL 2006 Problem Set #1 ANSWERS...

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Pset 1 KEY 1 7.06 FALL 2006 Problem Set #1 ANSWERS 1a. In order to better understand the function of the yeast protein Yup1, you are interested in learning where Yup1 localizes inside the cell using immunofluorescence (IF). In order to save your PI money, you decide to make a monoclonal antibody to Yup1 yourself, therefore you inject purified Yup1 protein into a mouse. After you have isolated the B lymphocytes from the spleen, what do you do to generate an antibody? The B lymphocytes must be fused with certain myeloma cells (myeloma cells are transformed B lymphocytes) and placed in selective media (HAT media) that will only allow fused cells to grow (by selecting against unfused B lymphocytes or unfused myeloma cells). HAT media is selective because the myeloma cells that you use lack HGPRT (an enzyme required for proper metabolism in HAT media). Unfused B lymphocytes will not grow for a long time in culture because they are an example of a primary cell culture that has a finite lifespan (a genetic transformation event has to occur first to make a cell line that can grow indefinitely). Fused cells will grow in HAT media because they have the HGPRT from the B lymphocytes needed for metabolism and the indefinite lifespan from the myeloma cells. These cells proliferate into hybridomas and must then be tested for specific reactivity to Yup1. Once the effective hybridomas have been identified, they are cultured to harvest large amounts of Yup1 antibody. b. Congratulations, you have successfully made a specific antibody to Yup1. Once you have made a slide with fixed yeast cells, how can you now use that antibody to visualize the localization of Yup1; will you need to use anything besides your antibody? Your anti-Yup1 antibody either has to be fused to a fluorochrome to be visualized by IF (called “direct IF”) or you have to use a secondary antibody (called “indirect IF.”) In this case you will need a secondary-antibody isolated from another organism that can bind mouse antibodies (for example, an antibody isolated from a goat that was injected with the constant region of mouse antibodies as an antigen). This secondary antibody has to be labeled with a fluorochrome to be detected using fluorescence microscopy. c. Based on the localization of Yup1 to the edge of the nucleus, you hypothesize that this protein might actually be residing in the nucleolus (a sub-compartment of the nucleus that houses the rDNA). How might you test that hypothesis and what reagents would you use to do so? You could show co-localization with an established nucleolar protein via IF. To do
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This note was uploaded on 01/23/2012 for the course BIOLOGY lsm1301 taught by Professor Seow during the Spring '11 term at National University of Singapore.

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706_F06pset1_ans - 7.06 FALL 2006 Problem Set #1 ANSWERS...

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