Laboratory Class 4: Lipids and membranes 2026
BCH 2333 Lab 4: Lipids and membranes Study Guide with Complete
Solution | 2026 Update
PROCEDURES
In experiment 1 you will prepare two membrane fractions from calf liver tissue: an endoplasmic
reticulum (ER) enriched fraction (microsomal fraction) and a mitochondrial enriched fraction. You
will extract the lipids from the microsomal fraction and separate them by TLC. A sample of
microsomal lipids will be provided so that you can start the TLC separation early in the lab session.
In experiment 2 you will analyze the lipid/ protein ratio of the mitochondrial fraction.
Before performing these experiments, you should watch the following videos:
Overview Homogenization (0:51) or (http://www.youtube.com/watch?v=NsYRQg40ZSQ)
Homogenization (3:34) or (http://www.youtube.com/watch?v=mZnLdYSV830)
Centrifugation (3:57) or (http://www.youtube.com/watch?v=5wCBHlAeoNw)
Rotovapour (2:42) or (http://www.youtube.com/watch?v=PPmITKfBwOo)
TLC (14:06) or (http://www.youtube.com/watch?v=IbU5l0DZD_Q)
You should also perform this simulation:
TLC: effect of solvent polarity
(https://elearning.cpp.edu/learning-objects/organic-chemistry/tlc/?page=simulation.html)
Finally, you should watch these animations:
Centrifugation
2D-TLC
The animations are located in the Laboratory 4: Lipids and membranes folder of the Laboratory
sessions module on the Brightspace site of the Laboratory component.
70
, Laboratory Class 4: Lipids and membranes 2026
EXPERIMENT #1: Lipids from the microsomal fraction of the liver a) Lipid
extraction
1. Mince 2.0g of calf liver tissue with scissors and place it in 10 mL of ice-cold
homogenization buffer (10mM Tris, 0.25M sucrose, 1mM EDTA, 1 mM DTT, pH 7.5).
2. Homogenize the tissue on ice with a glass-teflon homogenizer. Pass the pestle up and down
about 10 times. (Consult your TA).
3. Transfer the homogenate to a 15 mL Corex tube and centrifuge at 1,000xg for 10 min at
4°C. CAUTION! Tubes should be paired, and their weight balanced with homogenization
buffer.
4. Filter supernatant 1 through mira-cloth to remove lipid granules. Collect the filtrate in a 25
mL cylinder, measure its volume and transfer it into a new 15 mL Corex tube. Discard
pellet 1 which consists mostly of tissue debris, unbroken cells and nuclei (Figure 2).
5. Centrifuge supernatant 1 in the Corex tube at 25,000xg for 10 min at 4°C. CAUTION!
Tubes should be paired and balanced.
6. Transfer supernatant 2 into a 15 mL Falcon tube. This supernatant contains the microsomes
(Figure 2). Set aside pellet 2 for lipid and protein assay in experiment 2 (steps 22-27).
7. Transfer 3.2 mL of supernatant 2 from step 6 into a 50 mL Falcon tube and add 12mL of
CHCl3:MeOH (1:2 v/v). Mix well using a Vortex. (In the fume-hood!).
CAUTION! Chloroform and methanol are toxic, especially CHCl3; they
may irritate skin, eyes and respiratory tract. Use gloves and safety glasses.
Avoid inhalation.
(http://www.sciencelab.com/msds.php?msdsId=9927133
Methanol MSDS)
8. Centrifuge at 3000xg for 5min in a Sorvall Legend centrifuge (swinging bucket). (Tubes
should be paired!).
71
BCH 2333 Lab 4: Lipids and membranes Study Guide with Complete
Solution | 2026 Update
PROCEDURES
In experiment 1 you will prepare two membrane fractions from calf liver tissue: an endoplasmic
reticulum (ER) enriched fraction (microsomal fraction) and a mitochondrial enriched fraction. You
will extract the lipids from the microsomal fraction and separate them by TLC. A sample of
microsomal lipids will be provided so that you can start the TLC separation early in the lab session.
In experiment 2 you will analyze the lipid/ protein ratio of the mitochondrial fraction.
Before performing these experiments, you should watch the following videos:
Overview Homogenization (0:51) or (http://www.youtube.com/watch?v=NsYRQg40ZSQ)
Homogenization (3:34) or (http://www.youtube.com/watch?v=mZnLdYSV830)
Centrifugation (3:57) or (http://www.youtube.com/watch?v=5wCBHlAeoNw)
Rotovapour (2:42) or (http://www.youtube.com/watch?v=PPmITKfBwOo)
TLC (14:06) or (http://www.youtube.com/watch?v=IbU5l0DZD_Q)
You should also perform this simulation:
TLC: effect of solvent polarity
(https://elearning.cpp.edu/learning-objects/organic-chemistry/tlc/?page=simulation.html)
Finally, you should watch these animations:
Centrifugation
2D-TLC
The animations are located in the Laboratory 4: Lipids and membranes folder of the Laboratory
sessions module on the Brightspace site of the Laboratory component.
70
, Laboratory Class 4: Lipids and membranes 2026
EXPERIMENT #1: Lipids from the microsomal fraction of the liver a) Lipid
extraction
1. Mince 2.0g of calf liver tissue with scissors and place it in 10 mL of ice-cold
homogenization buffer (10mM Tris, 0.25M sucrose, 1mM EDTA, 1 mM DTT, pH 7.5).
2. Homogenize the tissue on ice with a glass-teflon homogenizer. Pass the pestle up and down
about 10 times. (Consult your TA).
3. Transfer the homogenate to a 15 mL Corex tube and centrifuge at 1,000xg for 10 min at
4°C. CAUTION! Tubes should be paired, and their weight balanced with homogenization
buffer.
4. Filter supernatant 1 through mira-cloth to remove lipid granules. Collect the filtrate in a 25
mL cylinder, measure its volume and transfer it into a new 15 mL Corex tube. Discard
pellet 1 which consists mostly of tissue debris, unbroken cells and nuclei (Figure 2).
5. Centrifuge supernatant 1 in the Corex tube at 25,000xg for 10 min at 4°C. CAUTION!
Tubes should be paired and balanced.
6. Transfer supernatant 2 into a 15 mL Falcon tube. This supernatant contains the microsomes
(Figure 2). Set aside pellet 2 for lipid and protein assay in experiment 2 (steps 22-27).
7. Transfer 3.2 mL of supernatant 2 from step 6 into a 50 mL Falcon tube and add 12mL of
CHCl3:MeOH (1:2 v/v). Mix well using a Vortex. (In the fume-hood!).
CAUTION! Chloroform and methanol are toxic, especially CHCl3; they
may irritate skin, eyes and respiratory tract. Use gloves and safety glasses.
Avoid inhalation.
(http://www.sciencelab.com/msds.php?msdsId=9927133
Methanol MSDS)
8. Centrifuge at 3000xg for 5min in a Sorvall Legend centrifuge (swinging bucket). (Tubes
should be paired!).
71
