Electron Transport Chain Inhibition
Name: (First and Last).
Date: DD/MM/YYYY format
Experiment #: 8
Title: Electron Transport Chain Inhibition
Purpose:
This experiment aims to introduce the use of mammalian cells and explore how structural changes can affect color changes and pH indicators. The experiment will further investigate how metabolism affects the cell’s acidification and how Electron Transport Chain (ETC) inhibition affects metabolism. The electron transport chain inhibition will be measured using colourimetric and pH methods. The experiment will help students understand the relationship between cell culture and acidification and the use of colorimetric and pH methods to measure inhibition.
Procedure:
- Preparation of mammalian cells
- Put phenol red in the well
- Pipette 100 microliters of cells into wells (25000 cells per well)
- Add 2ml of glucose
- Add Dimethyl sulfoxide (DMSO) to act as the control
- Addition of inhibitors
- Use 6 wells each with mammalian cells
- Add growth media to one well
- Add DMSO into two wells
- Add Antimycin (A) inhibitor to one well
- Add potassium cyanide, KCN (B), to one well
- Add both A and B to one well
- Qualitatively assessing colour/pH changes
- After day 1, day 4 and day 7, observe color changes and PH changes
- Below the PH 7 shows yellow color
- Above PH 7 shows the Red color
Data/Results/Calculations:
- Color changes
- Above the PH of 7 = Red (basic)
- Below the PH of 7 = yellow( acidic)
- But PH of 8 and 9 showed the color pink
- Dark yellow color at PH 5
Color change on Day 4.
| DMSO | Pink |
| A | More yellow |
| B | Reddish |
| A+B | Yellow |
Color change on Day 7.
| DMSO | Less pink |
| A | yellow |
| B | Reddish |
| A+B | Yellow |
- PH Analysis
- Plate 1 (Day 1) = All had the PH of 8.2 (Starting solution)
- PH of Plate 2 (After 4 days)
| Media | 8.0 |
| DMSO | 8.0 |
| A | 6.8 |
| B | 7.8 |
| A+B | 6.5 |
- PH of Plate 3 (After 7 days)
| Media | 7.8 |
| DMSO | 7.8 |
| A | 6.0 |
| B | 7.4 |
| A+B | 5.8 |
- UV/Vis Scan
- DMSO had the highest absorbance at 560nm (red)
- KCN (B) dropped a bit in terms of PH at 560nm
- Absorbance increased at 415 nm
Discussion Questions:
Which complexes did the Antimycin A, KCN, and other inhibitors target?
- Antimycin A – Targeted Complex 4
- KCN Targeted Complex 3
Why did the absorbance at 415 nm increase?
The absorbance at 415 nm increased because of pH change; pH affects the ionization state of the molecules in the sample.
Why did pH go down with the combinations of Antimycin A and KCN (A + B)?
The addition of Antimycin A and KCN to a solution can cause the pH of the solution to decrease because the two chemicals have an acidic property which lowers the pH of the solution.
Conclusion:
In conclusion, this experiment showed how mammalian cells could be used for studying the effects of structural changes on color changes of pH indicators. This experiment determined that cell culture and acidification can affect metabolism and electron transport chain inhibition and that this can be measured using colorimetric and pH methods. Through this experiment, students have gained a better understanding of the relationship between cell culture and acidification and the use of colorimetric and pH methods to measure inhibition. This experiment was, therefore, successful in achieving its purpose.
Notes:
Mammalian cells are commonly used in laboratory studies due to their similarity to human cells and the ease with which they can be grown and maintained in a controlled environment. They are also used to produce protein-based pharmaceuticals, develop gene therapy strategies, and study the process of tumorigenesis.
The electron transport chain (ETC) is an important process in both glycolysis and the TCA cycle. In glycolysis, the ETC is used to extract energy from glucose molecules in the form of ATP. In the TCA cycle, the ETC is used to further break down the pyruvate molecules and other glycolysis products to generate more ATP. The ETC also produces NADH and FADH2, which are used in the oxidative phosphorylation process to generate ATP. Thus, the ETC is essential for generating energy in both glycolysis and the TCA cycle.
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Question 
Instructions:
As you watch the video lab you should keep a lab notebook just as you would if you were personally conducting the lab. You should use your lab notebook to write your lab report which will need to be typed and submitted before you take the corresponding lab exam. The lab report template can be found here. Download here.
Electron Transport Chain Inhibition
Your lab report for Lab 8 should include the following information in order to earn full points for this assignment:
Purpose: (1 point) This section should be written in complete sentences using third person, and present or past tense. The purpose should include the goal of the experiment. If there is more than one part to the experiment you may summarize all goals in this section. This short description should make it clear what skills or knowledge should be gained from the completion of the experiment.
Procedure: (2 points) The procedure should be a step-by-step set of instructions that is detailed enough that someone could use it to replicate the experiment. This is very similar to a recipe in cooking. You can write this section using paragraphs, numbered lists, or bullet points however it should be written in complete sentences (third-person, present or past tense), and well-organized. If there is more than one part to the experiment, you can have multiple subsections in the procedure.
Be sure to include a procedure for:
Preparation of mammalian cells
Addition of inhibitors
Qualitatively assessing color/pH changes
Data/Results/Calculations: (2 points) In this section you should organize all the observations and data that were collected as part of the experiment, being sure to clearly label each measurement and/or part of the experiment. You may wish to construct a data table for a neat and professional-looking report. Please note that screenshots from the lab video of data are not permitted.
Be sure to include data/results/calculations for:
Color changes
pH analysis
UV/Vis Scan
Discussion Questions: (2 points)
Which complexes did the Antimycin A, KCN, and other inhibitors target?
Why did the absorbance at 415 nm increase?
Why did pH go down with the combinations of Antimycin A and KCN (A + B)?
Conclusion: (2 points) The conclusion section should be a well-developed paragraph (at least 6 sentences), separate from the Data/Results/Calculations section. It should be written in complete sentences (third-person, present or past tense) and should summarize the results of the entire experiment(s). You should emphasize what important concepts were explored, what the results showed, what conclusions can be drawn, and how the purpose of the experiment was accomplished.
Notes: The notes section is where you should put information that does not belong in other sections. This would include background information or anything else that you deem important that was not directly related to performing the experiment