Cellular Respiration Lab Report

BIO181L- F3:15

February 16, 2017

Mitochondria and Cellular Respiration

Introduction

Cellular respiration is how organisms derive energy for proper functioning, such as heating & cooling, and providing electrical impulses in our brains. The first step of this process is glycolysis, which is a series of reactions that extract energy from glucose by splitting it into two-three carbon molecules called pyruvates. Oxygen is not needed for this step, also known as the anaerobic process. During pyruvate oxidation, each pyruvate from glycolysis goes into the mitochondria. The mitochondria is where most ATP synthesis occurs. Pyruvate oxidation converts pyruvate into acetyl CoA, producing NADH and releasing one carbon dioxide molecule in the process. (Khan Academy) The citric acid cycle is a chain of multiple chemical reactions that helps in producing some ATP. In eukaryotes the citric acid cycle takes place in the matrix of the mitochondria. One turn of the citric acid cycle releases two carbon dioxide molecules and produces three NADH, one FADH2, and one ATP. This cycle goes around twice for each molecule of glucose that enters cellular respiration because there are two pyruvates made per glucose. (Khan Academy) The electron transport chain produces the bulk of ATP. This chain is composed of a series of proteins and organic molecules found in the inner membrane of the mitochondria. Electrons are transferred from one member of the transport chain to another in a series of redox reactions.This process requires oxygen, making it aerobic. As electrons move down the chain, energy is released and used to pump protons out of the mitochondria, forming a gradient. This is also known as oxidative phosphorylation. (Khan Academy) In this lab, students will be comparing the quantity of mitochondrial protein between the preparations of white and dark muscle from chickens. (Areda, Boyles, Francis, & Hite, 2016) The Bradford Assay test will be the determining factor of protein presence. The Bradford protein assay is used to measure the concentration of total protein in a sample. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue.  ("[Bio101] Bradford Protein Assay")

Hypothesis

The dark meat of chicken will have the more mitochondrial protein than the white meat, because dark meat uses more cellular energy.

Objectives

• Isolate and quantify mitochondria protein through the process of cell fractionation.
• Students should be able to differentiate between the three types of cellular respiration. Not only should each group member know the difference, but should also be able to explain the processes of each, including: aerobic respiration, anaerobic respiration, and fermentation.
• Students who are able to explain the processes, should also be capable of identifying the end result of cellular respiration.
• Tell about the relation between cellular respiration, and oxidation-reduction reactions. Describe what happens to the oxidation number during this process. Do the molecules gain or lose electrons? Tell why oxygen is needed for cellular respiration.
• Energy is required for the process of cellular respiration to occur. Students should be able to identify where this energy comes from, and what is needed to produce this energy.
• Students should be able to name and identify the internal components of the mitochondria. The site where oxidative phosphorylation takes place should be known.
• Students should use the scientific method to compose a lab report. The lab report should include an analyzation of the data collected from the lab.

Materials

Part 1: Isolation of Mitochondria from Chicken

• 10% preparation (in PBS) of dark chicken meat
• 10% preparation (in PBS) of white chicken meat
• Microtubes
• Homogenizer
• Microcentrifuge

Procedures

Part 1: Isolation of Mitochondria from Chicken

1. Using the Digital Tissue Homogenizer, grind 50 mL of the prepared dark meat, and 50 mL of the prepared white meat. Grind one substance at a time, making sure to wash homogenizer in between the preparations.
2. Label two 1.5 mL microtubes. Measure 1.5 mL of each homogenized meat sample, and add the substance to the tubes.
3. Place the tubes in the centrifuge. Be sure that the weight of the tubes is evenly distributed before turning it on. Set the centrifuge to 1,000 RCF for 10 minutes, and wait while it removes the debris from the meat samples.
4. Using a pipette, remove the supernatants, and transfer them to the second set of pre-labeled tubes. The pellets will not be needed, but make sure to observe them before throwing them away.
5. Place the new set of tubes into the centrifuge, making sure to balance the weight. Program the centrifuge for the same speed and time. This will remove the next layer of debris.
6. When the centrifuge is done, remove supernatants using the pipette. Place the supernatants into the third set of pre-labeled tubes. These pellets will not be needed either, but be sure to observe them before discarding.
7. Place the third set of supernatants into the centrifuge, balancing the weight. This time, set the centrifuge to 10,000 RCF for 10 mins, in order to separate and pellet the mitochondria.

Materials

Part 2: Protein Quantification Using Bradford Assay

• Preparations from mitochondrial isolation
• Microtubes
• THQ Digital Tissue Homogenizer (Vol. 0.25 milliliter [mL]–120 mL capacity)
• Pipette
• PBS buffer
• Bradford dye reagent
• Spectrophotometer
• Cuvettes
• Parafilm

Procedures

Part 2: Protein Quantification Using Bradford Assay

1. Use a pipette to transfer 196 microliters of PBS into the pre-labeled tubes.
2. Remove 4 microliters of the previously resuspended mitochondrial pellet, and 4 microliters into each tube. This creates a 1:50 dilution. Invert each tube 3-4 times. This will mix the two solutions together before it is transferred to the cuvettes.
3. Each group should gather 2 cuvettes, and label them A and B.
4. Use the pipette to add 1 mL of the Bradford dye reagent to cuvette A and B.
5. Pipette 20 microliters of each mitochondrial suspension into the corresponding cuvette, using a new tip for each sample.

-Complete this step quickly and efficiently, as the samples are time sensitive.

1. Each group should gather 2 small pieces of parafilm, and place 1 piece over each cuvette.
2. Invert each covered cuvette 3 times.
3. Set a timer for at least 5 minutes, and allow the cuvettes to sit at room temperature. Do not let them sit for more than 60 minutes.
4. Use the provided spectrophotometer to measure the amount of light that has been absorbed. Be sure to take the readings at 595 nanometers. Write down data in corresponding lab worksheet.

Data

[pic 1]

[pic 2]

[pic 3][pic 4]

Analysis