EXPERIMENT 13: Isolation and Characterization of Erythrocyte Day 1: Isolation of Erythrocyte Steps 1 through 6 of the Switzer & Garrity protocol (pages 220-221) have been performed by the TA. We will be using sheep blood rather than pig blood. Steps 7 and 8 will be performed by students, as written, ~1 hour before the start of lab in order to shorten the length of time in lab on Day 1. Only one group member needs to be present for these steps. Students should report to lab at ~12:45 pm. 7. Add 25 ml of hypotonic phosphate buffer to a 50-ml plastic centrifuge tube containing 5 ml of the erythrocyte suspension. Cap the tube and invert several times to mix. DO NOT VORTEX! 8. Balance your tube against that of another group and centrifuge and leave it there for the TA to begin the spin. The samples will be centrifuged at 20,000 x g for 40 min. Be sure to record the appropriate rotor position in your notebook. The membrane pellet at the bottom of the tube will be dark red in color and loosely packed. Therefore it is important that you remove your tube from the centrifuge as soon as possible after the centrifugation cycle is complete. Carefully remove the clear red supernatant (hemoglobin, cytoplasmic contaminants) with a transfer pipette. 9. Gently resuspend the membrane pellet in the centrifuge tube in 25 ml of hypotonic phosphate buffer. Mix well by inversion and centrifuge at 20,000 x g for 20 min. Carefully remove and discard the supernatant above the membrane pellet with a transfer pipette. Be careful when decanting the supernatant so as not to dislodge the membrane pellet. 10. Repeat step 9 two more times. Note the change in color of both the membrane pellet and the supernatant with each wash. 11. After the last wash step, remove as much of the supernatant as possible and gently agitate the tube to resuspend the loosely packed, milky-looking membrane pellet in the residual buffer at the bottom of the tube. After resuspending the membrane pellet, you may notice a more tightly packed (white or tan) pellet that contains denatured protein. Take care to ensure that you do NOT resuspend this pellet. At this point, you should have 2 to 4 ml of concentrated membrane solution. Transfer this solution to a 15-ml screw-cap conical tube. Label the tube with your name and record the exact volume of the membrane solution in your notebook. Store at 4 o C. Lipid Extraction 1. Transfer 0.8 ml of the membrane suspension into two separate 15-ml screw-cap conical tubes. Add 3 ml of chloroform:methanol (1:2 volume ratio) to each tube. Cap and swirl each tube vigorously with a Vortex mixer for 30 sec. Be sure that you have a minimum of 100 µl of membrane solution remaining in the original 15-ml conical tube that is NOT used in the extraction. You will need this to perform BioRad protein assays on Day 2. 2. Add 1 ml of chloroform to each tube, cap the tube, and swirl vigorously for 30 sec. 3. Add 1 ml of water to each tube, cap the tube, and swirl vigorously for 30 sec. 1
4. Separate the methanol:water phase (top) from the chloroform phase (bottom) in each tube by centrifugation at low speed (~500 x g, 5 min) in a tabletop centrifuge. You will see a small disk of white precipitate (denatured protein) at the interface between these two phases. If this disk extends down too far into the chloroform layer (more than halfway into the phase), add 0.1 ml of 0.1 M HCl, mix, and repeat the last low-speed centrifugation step. 5. Omit. It is not necessary to remove the upper phase following centrifugation. 6. With a clean GLASS Pasteur pipette, carefully puncture through the denatured protein disk and remove the chloroform layer at the bottom of each tube. Transfer the chloroform layer from both tubes to a single, preweighed 4-ml glass vial with a Teflon-lined screw cap. Take care not to transfer any of the denatured protein layer (white precipitate) to these glass vials. 7. Evaporate the combined chloroform phase containing the erythrocyte membrane lipids to dryness by passing a stream on N 2 (g) over the top of the vial in a fume hood. 8. When the lipids have been dried completely, weigh the vial again and determine the mass of lipid that you have extracted from the erythrocyte membrane. Record the mass of lipids in your notebook. 9. Based on the mass of lipid present in 1.6 ml of the membrane solution, determine the milligrams of lipid present in the entire erythrocyte membrane solution. 10. Resuspend your dried lipids in 1 ml of chloroform. Cap the vial tightly (chloroform is very volatile), label it with your name, and store it at room temperature. Day 2: Thin-layer Chromatography of Lipids 1. Obtain a silica-gel TLC plate that has been activated in a 100 o C oven for 5-10 min. Use a pencil to softly draw a straight horizontal line across the plate, 1.5 cm from the bottom. It is essential that you do NOT score through the silica gel to the plate underneath. Beginning 1.5 cm from the left side of the plate, make short vertical marks 1 cm apart, which intersect the horizontal line. There should be 8 marks to indicate the origins where you will spot lipid samples and standards. Below the mark in position #1, softly write your group initials. Below the marks in positions #2-7, write the abbreviations for the lipids standards that will be spotted at each position, as follows: 2) Sp, for sphingomyelin; 3) Ch, for cholesterol; 4) PC, for phosphatidylcholine; 5) PE, for phophatidylethanolamine; 6) PI, for phosphatidylinositol; 7) PS, for phosphatidylserine. Position #8 will be used by another group. 2. Remove 0.4 ml of your extracted lipids in chloroform prepared on Day 1 and place in a 1.5- ml microfuge tube. Dry the sample under a stream of N 2 gas, being careful not to blow the liquid out of the tube. Resuspend the lipids in 40 µl of chloroform. 3. Using your 20-µl pipettor, apply ~15 µl of the resuspended lipids at origin position #1. Keep the area of application small by repeated spotting and drying of 2-µl aliquots. Be sure that the spot dries completely with each application. Try to keep the sample spot as concentrated as possible (small diameter) to improve resolution and reduce diffusion. Remember that the samples are in a solution of chloroform, which will evaporate readily. This means you need to keep the caps closed on tubes containing any lipid sample or standard when not in use. 2
4. Using the same technique as before, spot 10 µl of each of the lipid standards, except sphingomyelin, at the designated origin positions on your plate. Spot 15 µl of the sphingomyelin standard to the appropriate position. Do not spot anything at position #8 of this plate. When you have finished spotting your plate, exchange plates with another group. Using your 20-µl pipettor, apply ~15 µl of the resuspended lipid sample at origin position #8. Be sure to spot as before using 2-µl aliquots. In this way, each group will have lipid samples spotted on 2 separate plates. You can facilitate complete drying between spotting by spotting 2 µl of each sample then starting over. 5. When all the spots have dried, place one plate containing lipid samples from 2 groups (origin side toward the bottom) in an ascending chromatography tank containing 1 cm of mobile phase solvent A (CHCl 3 :MeOH:28%NH 4 OH, 65:35:5). Place the 2nd plate containing lipid samples from the same 2 groups in a tank containing 1 cm of mobile phase solvent B (CHCl 3 :acetone:meoh:glacialacoh:h 2 O, 10:4:2:2:1). Be sure that the mobile phase is in contact with the plate, but that the origin containing your spotted samples lies above the level of the solvent). While your TLC plates are running, you may prepare for the phosphate assay and complete the cholesterol assay and the BioRad protein assay. 6. Allow the mobile phase to travel up the stationary phase until the solvent front is about 0.5 cm from the top of the plate (1-1.5 h). Remove the plate from the tank, mark the position of the solvent front with a pencil, and allow the plate to air dry in the fume hood for ~ 5 min. 7. Spray the plate with 3% cupric acetate in 8% phosphoric acid. Place the plate in a 100 o C oven and heat for 2-3 hours. The TA will remove the plates and make them available for photocopying during the next lab period. 8. Measure the R f values of the lipid standards: Distance traveled by sample spot R f = Distance traveled by solvent front Based on the R f values of the lipids present in your sample, what lipids appear to be present in the erythrocyte membrane? Based on the size and intensity of the spots present in your sample compared to those of the lipid standards, which lipid(s) are most abundant in the pig erythrocyte membrane? Preparation for Phosphate Assay 1. Transfer 20, 50, and 100 µl of your extracted lipid sample in CHCl 3 to three 4-ml glass vials with Teflon-lined screw caps. 2. Dry each of the samples under a stream of N 2 gas in the fume hood. 3. In a designated fume hood, add 0.5 ml of 70% perchloric acid to each vial and resuspend each of the dried lipid samples with gentle agitation. 4. Cap the vials tightly with a Teflon-lined screw cap and incubate in a 160 C oven for 5 hr. After cooling to room temperature, these solutions will be used on Day 3. 3
Quantitative Cholesterol Assay The extracted lipids that are currently dissolved in chloroform cannot be used directly in this assay, since the chloroform will denature the enzymes that produce the colored product. Turn on your spectrophotometer to allow it to warm up. 1. Transfer 150 µl of your extracted lipids in CHCl 3 into a 1.5-ml microfuge tube. Dry them under a stream of N 2 and resuspend the sample in 150 µl of isopropanol. 2. Set up the reactions described in the table below in 1.5-ml microfuge tubes: Component Blank 1 2 3 4 5 6 µl of 2 mg/ml standard -- 5 10 15 20 -- -- µl Lipids in isopropanol -- -- -- -- -- 25 100 µl Isopropanol 100 95 90 85 80 75 -- ml Cholesterol reagent 1.0 1.0 1.0 1.0 1.0 1.0 1.0 3. Mix by inverting the tubes. DO NOT VORTEX. 4. Incubate all tubes in a 37 C water bath for 10 min. 5. Transfer the entire contents of the Blank tube to a 1.5-ml visible cuvette and blank your spectrophotometer at 520 nm. Read and record the A 520 values for tubes 1-6 within 30 min of completion of the 10-min incubation. 6. Prepare a standard curve by plotting A 520 on the y-axis versus µg cholesterol on the x-axis for tubes 1 through 4. 7. Based on the absorbance readings of tubes 5 and 6, determine the mass of cholesterol (µg) present in these tubes. 8. Based on the volume of lipid sample added to each tube, determine the concentration of cholesterol (mg/ml) in your lipid sample in isopropanol. All values calculated in Step 7 that are in the range of the cholesterol standards should be included in an average value. 9. Based on the volume of lipid sample in CHCl 3 (150 µl) used to prepare the sample for the cholesterol assay, determine the concentration of cholesterol (mg/ml) present in your original lipid sample. 10. Using the molecular weight of cholesterol (386.7 g/mol), calculate the concentration of cholesterol (mm) in your entire membrane sample. BioRad Assay of Protein Concentration (replaces the Folin Protein Assay) 1. Prepare two series of 13x100 glass tubes (14 tubes total) containing the following volumes of protein standard solution (1 mg BSA/ml) + water: Component 1 2 3 4 5 6 7 µl H 2 O 60 55 50 45 30 15 0 µl BSA 0 5 10 15 30 45 60 4
Prepare two series of 13x100 glass tubes (6 tubes total) containing the following volumes of your membrane solution + water: Component 8 9 10 µl H 2 O 57 55 50 µl membrane solution 3 5 10 2. Add 3.0 ml of BioRad Dye Binding Reagent to each tube, mixing immediately. 3. Incubate for at least 10 minutes (no more than one hour; record the exact time). Set your spectrophotometer to 595 nm. Using a new cuvette, blank the spectrophotometer against the negative control tube (tube 1). Read and record the A 595 of each sample by transferring the contents of the round tube to the square cuvette. 4. Calculate the average A 595 value of duplicate trials for each standard. Prepare a standard curve by plotting the A 595 (dependent variable) on the y-axis versus the known protein mass per assay tube in µg (independent variable) on the x-axis. 5. Determine which of the tubes 8 to 10 gave A 595 values that lie in the linear portion of the standard curve. Average the A 595 values of duplicate trials for each of these samples (e.g., if the duplicates of #8 were in the linear portion of the standard curve, average these two A 595 values). Calculate the mass of protein for each of these (averaged) values from the standard curve. Calculate the final concentration of protein in your membrane solution by averaging the mg/ml concentration determined for each sample. 6. Based on the total volume of the entire membrane solution isolated on Day 1, how many mg of protein are present in the total membrane fraction? 7. Using the total mass of lipid in the membrane solution (calculated at the end of Day 1), what is the mass ratio of protein to lipid in the erythrocyte membrane? Day 3: Completion of Phosphate Assay If there is a heat block on your bench, turn it on and adjust the temperature to 110. Some groups will have to share heat blocks. 1. Prepare a series of phosphate standard solutions in 4-ml glass vials using a 1mM stock solution as outlined in the table below: Tube Volume of standard solution (µl) Volume of perchloric acid (ml) 1 -- 0.50 2 20 0.48 3 50 0.45 4 100 0.40 5 150 0.35 5
2. Add and mix the following reagents, in order, to these 5 standard vials as well as to the 3 vials containing your hydrolyzed lipid sample: 2.4 ml of water 0.1 ml of 5% ammonium molybdate in water 0.1 ml of amidol reagent (containing 2.4-diaminophenol) 3. Seal all 8 vials with a Teflon-lined screw cap and incubate at 110 C for 30 min in a heat block. 4. Allow the vials to cool to room temperature and read the absorbance of all of the solutions at 660 nm. Use the solution in vial 1 to blank your spectrophotometer. 5. Prepare a plot of A 660 versus µmol of phosphate standard and generate a standard curve from the data. Based on the A 660 of your 3 lipid samples, how many µmol of phosphate are present in each vial? 6. Based on the fact that there is a 1:1 ratio of phosphate to phopholipid, and considering the total volume of the lipid fraction recovered on Day 1, how many µmol of phospholipids are present in your extracted lipid sample? 7. Based on the average molecular weight of the four phospholipids (~780 g/mol), how many milligrams of phospholipids are present in your total extracted lipid sample? 8. Based on the number of mg and the number of µmol of cholesterol present in your total extracted lipid sample, what is the mass ratio and mole ratio of cholesterol:phospholipids in the erythrocyte membrane? 6