Quiz 9 Question Pool Fall 2023

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Temple University *

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3096

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Biology

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Feb 20, 2024

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Quiz 9 Question Pool 1. Describe in detail the assay you will do this week on the cellular fractions from last week. (5 points) This week I will be performing the fluorescence microscopy assay. Using the four fractions collected from last week’s lab, they will be pipetted on slides with equal amounts and dye mixture added on top. The dyes used are mitotracker, WGA, and DAPI. Then observed under the different filter systems of the fluorescent microscope to compare the intensity of the staining of the particulate matter in the slides 2. Fill in the following table so that all tubes have a final volume of 60 μl: (5 points) Assay Tube # Desired [BSA] (mg/ml) Volume of 1 mg/ml BSA (μl) Volume of phosphate buffer (μl) 1 = blank 0 0 60 2 0.2 3 0.4 4 0.6 5 0.8 6 1.0 3. What does the Bradford assay measure? (1 point) Measures protein concentration 4. Which dye does the Bradford assay use? (1 point) Blue G-250 5. To determine the total protein content in a particular fraction, what must you multiply by what? Hint: there are 3 quantities! (3 points) Volume, dilution of fraction, absorbance at 595 nm 6. a. What dyes will we use for fluorescence microscopy and what do they stain? (4 points) Mitotracker—stains mitochondria WGA—stains cell membrane

DAPI- stains DNA b. In the slides, the intensity of staining of particulate matter is important. (1 point) 7. What is enzyme activity? (2 points) Ability to convert substrate to product 8. What is the significance of total activity? (3 points) indication of organelle content and enzyme loss 9. What is the significance of specific activity? (3 points) It is a measure of purity, and it is obtained by dividing the activity per mL by the total protein concentration in mg/mL 10. In what units will we measure enzyme activity? (2 points) Enzyme units (U) We will measure activity in absorbance units per 30 minutes, (designated Activity*)/ml 11. Which 2 enzymes are we following and where is each found? (4 points) Succinate dehydrogenase; found in mitochondrial inner membrane Acid phosphatase; found in lysosomes 12. a. With which instrument will we measure the amount of product formed by the enzymatic reactions? (1 point) spectrophotometer b. What property must a substance have in order to be measured by the instrument in part (a)? (1 point) absorbance c. What is the measurable product of each enzyme reaction? (2 points) NBT; becomes purple when reduced PNP; yellow when reduced 13. For an enzymatic reaction, what should a blank contain? Hint: the answer is NOT “extra phosphate buffer”! (2 points) The substrate and the other controls/buffers added to all the other tubes

14. What substances will you add to each enzyme reaction to stop it? What do these substances do? (5 points) SDS is added to the succinate dehydrogenase which breaks apart its subunits and deactivate enzyme Cold Na3PO4 at pH 12 is added to acid phosphatase which prefers an acidic environment. 15. a. For succinate dehydrogenase, what volumes of enzyme go into the reactions? There are 2 different amounts, corresponding to 2 different dilutions. (2 points) One set gets 0.5 mL of the appropriate fraction, the other set gets 50 µL (added after 0.45 ml phosphate buffer). b. If after 30 mins you measure an absorbance of 0.65 for a particular fraction at the lesser dilution (greater amount of fraction used), what is the activity*/ml of that fraction? Show your work! (2 points) 0.65 Activity*/0.5 ml = 1.3 16. a. For acid phosphatase, what volumes of enzyme go into the reactions? There are 2 different amounts, corresponding to 2 different dilutions. (2 points) 1st set: 100 microliters 2nd set: 10 microliters b. If after 30 mins you measure an absorbance of 0.65 for a particular fraction at the lesser dilution (greater amount of fraction used), what is the activity*/ml of that fraction? Show your work! (2 points) 0.65 Activity /100 µl 1 ml/1000 µl = 0.065

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