Lecture 8 🔬

Week 8 (Oct 24): Mitochondria & Chloroplasts – Structure Chapter 11: pg. 637-651; 653 (Ion channels) - 661 Chapter 14: pg. 811-817, 861-868 Chapter 12: pg. 726-735 Chapter 14: pg. 843-844  Questions 1. How does confocal laser-imaging reduces blurry images...

Week 8 (Oct 24): Mitochondria & Chloroplasts – Structure Chapter 11: pg. 637-651; 653 (Ion channels) - 661 Chapter 14: pg. 811-817, 861-868 Chapter 12: pg. 726-735 Chapter 14: pg. 843-844  Questions 1. How does confocal laser-imaging reduces blurry images confocal laser scanning only allows light from the exact focal plane to be captured, preventing light from areas above or below the focus point from interfering, thus reducing blurry images 2. What is the significance of using electrophoresis in protein analysis Electrophoresis is significant in protein analysis because: - It allows proteins to be separated based on their sizes - SDS will give all the proteins a uniform negative charge, making them linear (not folded) - It allows visualization for protein size and purity - Confirms correct separation occurs for further studies or applications 3. Explain the process of cell fractionation and its goal in protein purification Cell fractionation is the process of breaking open cells to release their content (proteins, organelles) to create a cell extract. The cell extract then allows us to separate the cellular components. The goal in protein purification is to isolate the proteins using several methods like centrifugation and chromatography. These methods allows the proteins to be separated based on size, charge or other properties for further studies. 4. What are the differences between differential centrifugation and density gradient centrifugation Differential Centrifugation Density Gradient Centrifugation Spins the extract at increasing Uses a sucrose gradient speed Heavier parts settle first (nuclei) Components will move at different speeds, forming distinct “bands” in gradient Looks at the mass of the Looks at the size of the components components 5. How does column chromatography separate proteins based on their properties? - gel filtration chromatography separates proteins by their size. the column is packed with beads that have tiny holes in them. the big proteins will not be able to pass through the hole so they go through the column faster. the smaller proteins will get stuck to the holes and take a longer time to move through the column. - ion-exchange chromatography uses the charge of proteins to separate them. a charged column with either a positive or negative charge will attract proteins with the opposite charge. by changing the salt or ph of the solution, the proteins can be released from the column at different times. - affinity chromatography separates proteins based on how well the proteins bind to specific molecules. the column is coated with a substance like a protein or antibody that only binds to protein of interest. other proteins will just pass through without binding while the target protein will bind and can be collected separately.  Dr. Smith is conducting an experiment using Confocal Laser-Scanning Microscopy to study the distribution of a fluorescent protein tagged with GFP in live cells. The setup includes a laser beam directed at the sample, and a pinhole aperture is used to minimize out-of-focus light. During the imaging process, Dr. Smith notices that background noise is affecting image clarity. 6. What advantage does the use of a pinhole aperture provide in Confocal Laser-Scanning Microscopy? The pinhole aperture only allows light from a specific part of the sample or focus plane to reach the detector 7. If Dr. Smith wants to reduce background noise further during imaging, which microscopy technique could he consider implementing? TIRF Microscopy. TIRF Microscopy shines light at an angle that only illuminates a thin layer of the sample, which minimizes the background light (noise) and allowing a clearer image of individual molecules. 8. How does confocal microscopy enhance image clarity? By using a pinhole to focus on a specific plane 9. What advantage does multi-photon microscopy have over traditional confocal microscopy? Deeper imaging with less damage to the sample 10. How does multi-photon microscopy reduce damage during imaging? By using lower-energy infrared light Dr. Smith is conducting an experiment to track the movement of a newly discovered protein in live cells. He decides to use GFP technology, attaching the gene for GFP to the gene encoding the protein of interest. He also plans to utilize FRAP to observe how the protein behaves over time. However, he is concerned about background noise affecting his results during imaging. 11. What advantage does using GFP technology provide in Dr. Smith's experiment? GFP is made out of jellyfish protein that can glow green under certain light. It can be attached to a gene of a protein that they want to study, which allows the cell to make a combined protein. this technique allows us to locate the specific proteins, track them and observe their behaviour. 12. What does Dr. Smith expect to observe in the FRET analysis when proteins A and B interact closely? A decrease in fluorescence from one protein and an increase in fluorescence of the other. 13. If Dr. Smith observes rapid recovery of fluorescence after bleaching in her FRAP experiment, what can be inferred about the mobility of the proteins within the cell? The proteins are highly mobile and able to move into the bleached area quickly, suggesting dynamic interactions. 14. How do super-resolution techniques like STORM enhance imaging capabilities compared to conventional microscopy? STORM is able to take pictures at much higher resolutions than microscopes, which allows scientists to see details at the nanometer scale (which is way smaller than the resolution limit of conventional light microscopes)  A researcher is studying a novel protein involved in cellular signaling. To isolate this protein, she begins with cell fractionation using mechanical disruption followed by differential centrifugation. After obtaining the supernatant, she applies size-exclusion chromatography to separate proteins based on their size. She notes that her target protein elutes later than smaller proteins. 15. What is the purpose of using differential centrifugation in the initial steps of protein isolation? To separate cellular components based on size and density, allowing for the collection of the supernatant containing soluble proteins. 16. Why would size-exclusion chromatography be an appropriate method for purifying the target protein after centrifugation? It separates proteins based on size, allowing larger proteins to elute first while smaller proteins are retained in the column, facilitating the isolation of the desired protein. In a laboratory, researchers are tasked with isolating a specific protein from a complex mixture. They decide to use both chromatography and SDS-PAGE for their experiments. Initially, they perform ion-exchange chromatography to separate proteins based on charge. After fractionation, they analyze the collected fractions using SDS-PAGE to determine the size of the target protein. 17. What is the primary principle behind ion-exchange chromatography used in this scenario? Ion-exchange chromatography separates proteins based on their charge by utilizing a charged column that attracts oppositely charged proteins. 18. Why is SDS added during the electrophoresis step in SDS-PAGE? SDS is added to give all the proteins a uniform negative charge and make them linear (not folded @ denatured). This allows the proteins to be separated based solely on size. 19. How does ion-exchange chromatography release bound proteins? By changing the pH or salt of the solution. 20. In the initial step of gel filtration chromatography, which type of proteins would elute first and why? The larger proteins will elute first because gel filtration chromatography uses columns that is packed with small beads that has tiny holes. The smaller proteins will be stuck in the holes thus elute later than the larger proteins which will not be able to fit through the tiny holes, thus just passes through. 21. What is the primary mechanism behind ion-exchange chromatography for separating proteins? This method uses the charge of the proteins to separate them. The column will have either a positive or a negative charge on it, and will attract proteins with the opposite charge. The bounded proteins can be released from the column by changing the pH or the salt of the solution used in the column. 22. How can the biochemist effectively elute the bound protein from the affinity chromatography column? By using a solution with free antibodies or antigens to release the proteins. 23. What primary factor does SDS-page utilize for protein separation? Size of proteins.