L4 Proc

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Apr 3, 2024

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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.

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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

Laboratory Class 4: Lipids and membranes 2024 74 16. Place the plate in a tank containing the basic solvent system: CHCl 3 -MeOH-ammonia (65:35:5 v/v). Develop the first dimension until the solvent reaches 1 cm from the horizontal line (about 60 min). 17. Remove the plate, mark the solvent front immediately and dry with a heat gun for 10 minutes. 18. Turning the plate 90 degrees, place it in the second tank with the acidic solvent system: CHCl 3 -acetone-MeOH-acetic acid-H 2 O (10:4:2:2:1; v/v). Develop the second dimension until the solvent reaches 1 cm from the horizontal line. 19. Remove the plate, mark the solvent front immediately and dry the plate with a heat gun. 20. Place the plate in an iodine saturated tank for 5 min. Remove the plate, leave it in the fume-hood and mark the position of each spot. Read the position of each spot with the plate ruler. 21. Your TA will scan your TLC plate. Your file will be provided in your dataset. Add the appropriate legend to this figure and submit it with your report. EXPERIMENT #2: Assay of mitochondrial lipids and proteins 22. Resuspend the pellet from step 5 in 10 mL of homogenization buffer. Sonicate the suspension at 50% power for 10 sec, twice, to accelerate the suspension process. This suspension contains the mitochondrial membranes . 23. Calculate the missing values ( ? ) in Table 2 and 3; this should be completed prior to your arrival in the lab. Verify your calculations with your TA before starting the experiment. CAUTION ! Iodine is harmful for man and the environment. Avoid inhalation and contact with skin. Do not leave the iodine tank open, wear gloves. ( http://www.sciencelab.com/msds.php?msdsId=9927547 )

Laboratory Class 4: Lipids and membranes 2024 75 24. Prepare five assay tubes containing increasing amounts of the mitochondrial membrane suspension in 2 mL of 0.1 M (final concentration) phosphate buffer, pH 11, according to Table 2 . Upon addition of the last reagent, the dye Brilliant Blue R, vortex thoroughly and incubate for 15 min at room temperature. (During this incubation, start the protein assay, step 26). Table 2: Lipids assay Sample Dilution fraction Phosphate pH 11, 0.2M (mL) Water (mL) Membrane suspension ( L) Brillant blue R 1.0g/L ( L) A 559 1) Control ? 1 0.95 0 50 2) 1/250 ? 1 ? 8 50 3) 1/125 0.008 1 ? 16 50 4) 1/80 ? 1 ? 25 50 5) 1/25 ? 1 ? 80 50 25. After incubation, read the absorbance at 559 nm using test tubes, zeroing the apparatus with water. Record these readings on Table 2 . 26. Prepare four assay tubes containing 1/4, 1/25 and 1/100 dilutions of the mitochondrial membrane suspension, and a blank, in 2 mL of 0.1 M phosphate buffer, pH 11, according to Table 3 . Table 3: Protein assay 27. Mix well and read the absorbance at 280 nm (in order of increasing concentration) using a 1 mL plastic cuvette (transparent to UV). Zero the apparatus with . Record these readings on Table 3 . Sample Dilution fraction Phosphate pH 11, 0.2M (mL) Water (mL) Membrane suspension (mL) A 280 1) Blank 0 1 1 0 0 2) 1/100 0.01 1 ? ? 3) 1/25 ? 1 ? ? 4) 1/4 ? 1 ? ? CAUTION! Brilliant Blue R solutions may cause eye, skin, and respiratory tract irritation and it will dye your skin. Use gloves and safety glasses. ( http://www.sciencelab.com/msds.php?msdsId=9925778 )

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Laboratory Class 4: Lipids and membranes 2024 76 RESULTS AND DISCUSSION Two-dimensional TLC of microsomal lipids R1. Attach a figure of your TLC plate with corresponding labels for all the lipids. Prepare a summary table providing the Rf values of the lipid standards for each of the two solvent systems. Are the Rf values of all standards identical for both solvent systems? If not, explain why some differences were observed. R2. From your results, rank the standard lipids by increasing order of polarity for each of the two chromatographic conditions that were assessed. Include the chemical structures of all lipid standards and, for each one, identify the polar groups. For each pair of standards, compare the polarity ranking within members of the pair. Explain your reasoning. Lipid and protein assay of mitochondrial suspension R3. For each assay prepare: a) a table containing the dilution fraction and the absorbance. b) a plot of the absorbance at 559 nm (lipid assay) and at 280 nm (protein assay) against the dilution fraction, including a point for the blank (reagent alone, no sample). In the protein assay plot, your control sample should be at zero. For the lipid assay plot, the linear region of the plot is normally found within the three or four most dilute samples. These slopes represent the decrease/increase in absorbance corresponding to a dilution factor of one, that is, the undiluted sample. Remember that the slope is equal to Y/ X or (Y 2 -Y 1 )/(X 2 -X 1 ), if you are using the values from the reagent alone reaction (0, 0), as well as the values from the reaction using the undiluted sample (1, Y), you will get the following relation: Slope = (Y-0)/(1-0) where Y correspond to the absorbance of the undiluted sample.

Laboratory Class 4: Lipids and membranes 2024 77 R4. Assuming an absorptivity (or extinction coefficient) at 280 nm of = 2.0 Lg -1 cm -1 for membrane proteins, calculate the protein concentration of your membrane preparation from the increase in absorption corresponding to your undiluted sample. Show your calculation. R5. Assuming that 1.0 g/mL of membrane lipids causes a decrease in Brilliant Blue R absorption at 559 nm of 0.013, calculate the concentration of lipids in the mitochondria- enriched fraction from decrease in absorption corresponding to your undiluted sample. Show your calculation. R6. From your results, estimate the yield for your mitochondria preparation in mg of mitochondrial proteins per gram of liver tissue (mg/g).

Laboratory Class 4: Lipids and membranes 2024 78 REFERENCES 1. Bligh, E.G. and Dyer, W.J. (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911. 2. Kates, M. (1986), Techniques in Lipidology. Revised 2nd edition. Elsevier Bio-medical Press. (Amsterdam, New York), pp 349, 428, 552 and 553. 3. Voet, D. and Voet, J. (2004). Biochemistry, 3rd Ed. John Wiley & Sons, p 152. 4. Baenziger, J. (2004). Ultracentrifugation.Tutorial notes VI. 5. Voet, D. and Voet, J. (2004). Biochemistry, 3rd Ed. John Wiley & Sons, p 382 and 394. 6. Boyer, R.F. (2000). Modern Experimental Biochemistry, 3rd edition. Benjamin Cummings, pp 61 and 357. 7. Bevilacqua, L. and Rodriguez, M. A. (2006). Chromatography. Tutorial notes V. 8. Voet, D. and Voet, J. (2004). Biochemistry, 3rd Ed. John Wiley & Sons, 6.3, p 133.

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