Problem Set 3

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Feb 20, 2024

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Problem Set 3 Principles of Cell Biology Fall 2022 1. There are many occasions when as a cell/molecular biologist you need to separate cells bound together in either a tissue or a cell culture so that they can be sorted and analyzed. The following questions relate to this statement. a. What is the purpose of EGTA and protease in this process and how does each work? EGTA and protease are both required for cell separation to occur. EGTA is a calcium chelator which works be disabling calcium dependent cell adhesion molecules. Proteases like trypsin and collagenase work by cleaving proteins. b. Challenge : You need to select which cell separation technique to use in an experimental protocol. Your laboratory has a FACS machine, can do velocity sedimentation, and also has magnetic beads such as Dynabeads. How would you decide which cell separation technique to select? If the cells don’t differ in physical size, then the magnetic beads would have to be used as velocity sedimentation can only work when there are differences in cell size. c. Challenge : You do an experiment that requires the use of Annexin V and Propidium iodide as described in class to determine the number of cells undergoing apoptosis in a culture exposed to a chemotherapy drug candidate. Why is this assay an appropriate one to use in this case and how could you determine using a non-microscope technique the percentage of cells in the entire population that are undergoing apoptosis compared to those not undergoing apoptosis? Because the chemotherapy drugs are causing the cells to undergo apoptosis using Annexin V and Propidium is appropriate since these dyes are able to bind to and fluoresce cells that are in the beginning and late stages of apoptosis. In order to count the number of cells that are undergoing apoptosis it is sufficient to count to number of fluorescent cells. This can be done using FACS or Guava. d. The Veridex system can also enumerate cells as well but is designed to count Circulating Tumor Cells (CTCs). How does it work and why is counting CTCs an important future clinical tool? The Veridex system works by targeting a protein that is found uniquely Circulating Tumor Cells and no where else. This protein is bound to by a antibody carrying a piece of iron which can then be detected and quantified. Counting CTCs is important because it can quantify “Tumor Burden” and can show how harmful a tumor is to the patient. 2. You decide to study a lysosomal storage disease (there are 40+ of them some of which we will detail later in the semester) exhibited by a child and realize that you need to purify lysosomes from a tissue biopsy of this child in order to further analyze the molecular basis of this disease. The biopsy you select is harvesting some nucleated blood cells which is the most non-invasive approach that can be used. The following questions relate to this research problem. a. You realize that the first step in the process would be to purify lysosomes using some type or series of centrifugation process(es). You select differential centrifugation as your starting point. Why is this a good choice?

By using differential centrifugation, we are able to separate a sample into discrete parts sorted by weight. By using a known RMP it is possible to separate liposomes, and everything with around the same mass as a liposome, from the rest of the tissue biopsy. b. Once you complete the above step then you need to now move to Rate Zonal centrifugation to further enrich the lysosomal fraction. Why is this the case? After differential centrifugation everything with around the same mass as a liposome is collected. Unfortunately, this also includes Mitochondria and Peroxisomes. By using Rate Zonal centrifugation, we can separate this mixture since these things have different densities. c. Using Rate Zonal you could also purify rough microsomes from smooth microsomes which, albeit not relevant to this lysosomal research project, is of general interest. What is the difference between rough vs smooth microsomes? Rough microsomes originate from the rough endoplasmic reticulum. Smooth microsomes are mainly from the cell membrane. 3. As has been evident in class a variety of probes are available to use to address a variety of cell biology questions. Two such probes discussed in class are photo-leucine and 3 H- leucine. How do they differ from each other in their applications in the research lab? Photo-leucine is photo-reactive while 3 H-leucine is radioactive. 4. There are a number of techniques to separate and/or analyze proteins. The major ones were discussed in class. Match the technique to each of the descriptions below. a. You are separating out the LDL receptor by using a column chromatography system that uses a mAb directed against the LDL receptor. Affinity Chromatography b. You are developing a protein blood biomarker assay that requires you distinguish 3 different related proteins that are identical in amino acid sequence except that the 3 proteins differ from each other in only their respective molecular weight by one and two amino acids respectively from the parent molecule. SELDI-TOF c. You are separating proteins in a gel that is characterized by a pH gradient that ranges from 3 to 10. Isoelectric Focusing d. You are using a technique that combines isoelectric focusing with SDS gel electrophoresis into a single technique. 2-D Gel electrophoresis e. You are working with nuclear proteins isolated initially from the nuclear fraction harvested via differential centrifugation. You are attempting to isolate a 300,000 Dalton protein from all of the other proteins that are less than 20,000 Daltons and you select a process that separates proteins based on physical size. Gel Filtration f. You are separating denatured proteins by first boiling your sample in the presence of urea and beta mercaptoethanol. SDS Gel electrophoresis

g. You are doing an experiment whereby you want to measure the relative abundance of a protein in a control versus an experimental using a gel system that incorporates a mAb and requires the use of a transfer membrane Western Blot h. You are using positively charged beads packed into a column to separate proteins that exhibit differences in their respective overall negative charges so that you can measure the enzymatic activity of the isolated protein fraction in question. DEAE ion exchange chromatography

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