L4 Proc

.pdf

School

University of Ottawa *

*We aren’t endorsed by this school

Course

2333

Subject

Biology

Date

Apr 3, 2024

Type

pdf

Pages

9

Uploaded by MegaHorsePerson382

Laboratory Class 4: Lipids and membranes 2024 70 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.
Laboratory Class 4: Lipids and membranes 2024 71 EXPERIMENT #1: Lipids from the microsomal fraction of the liver a) Lipid extraction 1. Mince 2g 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 CHCl 3 :MeOH (1:2 v/v). Mix well using a Vortex. (In the fume-hood!) . 8. Centrifuge at 3000xg for 5min in a Sorvall Legend centrifuge (swinging bucket). ( Tubes should be paired! ). CAUTION! Chloroform and methanol are toxic, especially CHCl 3 ; 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 )
Laboratory Class 4: Lipids and membranes 2024 72 9. Transfer supernatant to a new 50 mL Falcon tube. (Discard the pellet). Add 4 mL of CHCl 3 and 4 mL of water. Mix well using the Vortex. (In the fume-hood!) . CAUTION! All handling of chloroform in this lab should be done, when possible, in the fume-hood, using proper gloves. 10. Centrifuge at 3000xg for 5 min using the Sorvall Legend centrifuge (swinging bucket). ( Tubes should be paired! ) 11. Using a transfer pipette, transfer the lower phase into a 100 mL round bottom flask. (Do not take any liquid from the top phase). 12. Evaporate the chloroform in a rotary evaporator. (Consult your TA). Dissolve the residue in 0.5 mL CHCl 3 and transfer to a 1.5 mL microtube. Label the tube with the date, your lab section and group number, and hand it to your TA. Total lipids can be extracted in a similar way using the liver homogenate from step 2. Alternatively you can perform the tissue homogenization in CHCl 3 :MeOH (1:2 v/v). By adding 1/3 volume of water and 1/3 volume of CHCl 3 the mixture separates in two phases. As before, the bottom phase (CHCl 3 ) contains the lipids. b) Two-dimensional TLC 13. Each student will perform a 2D-TLC separation of a microsomal lipids sample. The sample will be provided at the beginning of the lab session so that you can start the TLC separation right away and finish it within the lab period. 14. Label an activated (heated at 100°C for 1-2 hours) 20 x 20 cm Silica G plate using a TLC template and a pencil. Mark only the spots, lines and traveling direction of the two developing solvents as seen in Figure 3 . On the top left corner label your plate with the date, your sections and group numbers.
Laboratory Class 4: Lipids and membranes 2024 73 15. Pipet 5 L of the standards and 10 L of the sample at the positions indicated in Figure 3 . Keep the spots as small as possible to increase resolution. Lipid standards: 1) CL (cardiolipin) and CH (cholesterol) 2) FA (fatty acid) and PC (phosphatidylcholine) 3) PI (phosphatidylinositol) and PE (phosphatidylethanolamine) Which standard in each couple will run furthest? Figure 3. TLC Template. Schematic depicting the labeling and loading of the 2D-TLC plate for experiment 1. 3 2 1 3 2 1 standards 2.5 cm sample standards 5 cm 5 cm 2.5 cm ID 2 nd dimens. 1 st dimens
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
  • Access to all documents
  • Unlimited textbook solutions
  • 24/7 expert homework help