Cell S&F Lab Quiz 9

1. Describe in detail the assay you will do this week on the cellular fractions from last week. (5 points)

-We will perform a Bradford assay to measure protein concentration of the fractions. -We will use brilliant Blue G-250 dye to bind proteins. -The proteins will be detected at 595 nm wavelength using a spectrophotometer -Then proteins can be quantified using Beers Law.

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) 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)

1st set: 100 microliters 2nd set: 10 microliters 0.65 Activity*/100 µl * 1 ml/1000 µl = 0.065

7. What is enzyme activity? (2 points)

Ability to convert substrate to product

b. What property must a substance have in order to be measured by the instrument in part (a)? (1 point)

Absorbance

4. Which dye does the Bradford assay use? (1 point)

Brilliant Blue G-250

3. What does the Bradford assay measure? (1 point)

Concentrations of protein

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.

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.

8. What is the significance of total activity? (3 points)

It measures the total amount of enzyme in the fraction Significance: indication of organelle content and enzyme loss

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

c. What is the measurable product of each enzyme reaction? (2 points)

NBT; becomes purple when reduced PNP; yellow when reduced

4. 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.

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

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

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, and absorbance at 595 nm.

b. In the slides, the intensity of staining of _______________________________ is important. (1 point)

particulate matter

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) 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)

s) One set gets 0.5 mL of the appropriate fraction, the other set gets 50 µL (added after 0.45 ml phosphate buffer). 0.65 Activity*/0.5 ml = 1.3

12. a. With which instrument will we measure the amount of product formed by the enzymatic reactions? (1 point)

spectrophotometer