Assignment: Cell Fractionation Worksheet
Assignment: Cell Fractionation Worksheet
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All questions are listed. All data is listed. This was a pre-preformed lab.
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Worksheet for cell Fractionation, Chapter 8
Introduction
Cellular fractionation is a technique that enables researchers to isolate cellular organelles. Once isolated, the presence and functions of these organelles can be studied biochemically.
- Characterization of Mitochondria by Succinic Dehydrogenase Assay
The presence of mitochondria is measured by the ability of the fraction to metabolize succinate and reduce DCPIP (blue) to DCPIPH2 (clear).
Procedure: Described in Powerpoint and in Teams recording
Table1.
Sample (Fraction) 1 | Sample (Fraction) 2 | |
At time t = 0 | blue | blue |
After 15 minutes | 0.8 mm clearing | 1.2 mm clearing |
- Which sample (fraction) contains higher concentration of mitochondria? Explain why. (4 points)
- Characterization of Mitochondria by Differential Spectral Analysis
Procedure: Described in Powerpoint and in Teams recording
Results:
Table 2: absorbance values of scan of fractions 1 and 2.
nm | Fraction 1 | Fraction 2 |
400 | 0.02 | 0.02 |
425 | 0.08 | 0.2 |
450 | 0.15 | 0.45 |
475 | 0.42 | 0.25 |
500 | 0.85 | 0.15 |
525 | 0.38 | 0.08 |
550 | 0.42 | 0.15 |
575 | 0.26 | 0.16 |
600 | 0.25 | 0.17 |
625 | 0.18 | 0.18 |
650 | 0.15 | 0.5 |
675 | 0.16 | 0.65 |
700 | 0.18 | 0.02 |
725 | 0.2 | 0.05 |
- Graph the data above on the same axis with Absorbance on Y-axis ( points) and wavelength on the X-axis. (4 points)
- What is the role of CaCl2 used in the spectral analysis? (2 points)
Note: Since the absorbance is directly proportional to the concentration, we will also these abs as concentrations in mM.
- Use the graph and determine the concentrations of CytC; CytC reductase, and CytC oxidase and complete table 3 (6 points)
Table 3. Abs values (or mM)
Abs (concentration) mM | Concentrations in Fraction 1 | Concentrations in Fraction2 |
Cytochrome C (505 nm) | ||
Cytochrome C reductase (562 nm) | ||
Cytochrome C oxidase (605 nm) |
- Which form of cytochrome C is more predominant in fraction 2? (2 points)
- Attended the lab? _____________ (2 points)
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