Lab 5 - Flagellar Isolation from Chlamydomonas

Biology 29 Cell Structure and Function Spring, 2009 Lab 5 - Flagellar isolation from Chlamydomonas Introduction: In this laboratory you will be using biochemical extraction procedures and high speed centrifugation to isolate flagellar proteins from the unicellular green alga Chlamydomonas reinhardtii. At various stages during this procedure, samples will be saved to be used in the second part of the experimental protocol: electrophoretic separation of proteins. For background on cell fractionation and differential centrifugation, see your text. Procedures: Isolation Students will work in teams to isolate flagellar proteins, as well as to carry out SDS polyacrylamide gel electrophoresis. 1. Three groups will start with one 1 L culture of Chlamydomonas. The cells will be evenly divided between four 250ml centrifuge bottles and centrifuged in the superspeed centrifuge (large rotor) at 3000 rpm (1300 x g) for 6 minutes at room temperature. Each group will receive one bottle and should work independently from this point onward. Discard the supernatant (medium) from the centrifuge bottle by immediately and CAREFULLY pouring most of the supernatant into the sink near the centrifuge. Do NOT pour quickly, or shake the centrifuge bottle, as the cell pellet is very soft and easily dislodged. 2. After decanting most of the supernatant, remove as much of the remaining supernatant as possible by aspiration (the lab instructor will demonstrate how to do this). Since the pellet is soft, it will be easy to aspirate and you must exercise caution not to suck up cells as well as medium. At this point it is more important to retain as many cells as possible than to get the pellet absolutely dry. 3. After aspirating the supernatant, resuspend the cells (pellet) in 10 ml of 10 mM HEPES buffer, pH 7.4. This is best done by resuspending the pellets by trituration (this consists of repeatedly sucking the pellet up and blowing it out with the pipet; the instructor will demonstrate this), and then pooling them in a single 30 ml centrifuge tube. 4. Each group should save a 1 ml aliquot of the culture to be used later for protein analysis and electrophoresis. This sample should be placed in a microfuge tube and kept on ice. 5. Spin the cells, being sure to balance the centrifuge, for 8 minutes at 3200rpm (1300 x g) using the small rotor. 6. Remove the supernatant by aspiration and resuspend the washed cells in 5 ml of ice cold HMDS (10 mM HEPES, 5 mM MgSO4, 1 mM DTT, 4% sucrose, pH 7.4). From here on the samples should be kept cold at all times. This means that both your solutions and your centrifuge tubes should be kept on ice. 7. Add 0.5 ml of 50 mM dibucaine to the cells (final concentration: 5mM) and immediately mix by swirling. Dibucaine is a drug (used as a local anesthetic) which causes the cells to lose their flagella. After addition of dibucaine, a sample of the cells should be observed with the phase contrast microscope to be sure they are really deflagellated. If flagella are still attached to the cells, add another 0.5 ml of dibucaine and check again. 8. Add 5 ml ice cold HMDES (HMDS containing EGTA, aprotinin, and PMSF 1), to your cell suspension and mix gently, but thoroughly. (final concentrations: 2 mM EGTA, 5 mM aprotinin, and 1 mM PMSF). Gently mix the cells with a 5 ml pipet, then transfer this solution to a chilled 16 ml round bottom tube. 9. Spin the cells in the small rotor at 4000 rpm (2000 x g) for 5 minutes at 4oC. 1 10. Using a 10 ml plastic pipet, carefully transfer the supernatant to a clean tube and repeat this spin (5 min., 4000 rpm) to remove any remaining cell bodies. 11. Save the pellet from the first spin, which contains the cell bodies, to use for the second half of the experiment (resuspend the pellet in 2 ml of ice-cold HMDEK [30 mM HEPES, 5 mM MgSO4, 1 mM DTT, 0.5 mM EGTA, 25 mM KC2H3O2, 5 mM aprotinin, 1 mM PMSF, pH 7.4] and keep it on ice). 12. Using a 10 ml plastic pipet, carefully transfer the supernatant, which contains the flagella, into a clean, chilled 16 ml tube. Spin the tubes containing the supernatant at 10,000 rpm (12,000 x g) for 12 minutes at 4oC to pellet the flagella. 13. Carefully remove the supernatant from the flagellar pellet by aspiration (the instructor will demonstrate). Resuspend the flagella in 250 μl HMDEK. Note: EGTA is a divalent metal ion chelator which helps to inhibit proteolysis during the extraction procedure; aprotinin and PMSF are protease inhibitors; DTT (dithiothreitol) is a reducing agent to prevent oxidation of proteins. Protein assay: Run a protein assay on samples of the following: whole cells, deflagellated cells, isolated flagella The purpose of this assay (or test) is to determine the concentration of protein in your various samples, so you can determine appropriate volumes for loading onto an SDS polyacrylamide gel in next week's lab. What you will do is to run the protein assay on both your own samples and a set of "standards" of known protein concentration. This set of known concentrations will be used to plot a standard curve which will allow you to determine the protein concentration of your samples. The standard curve is plotted based on knowing two dependent parameters for the standards, that is (1) the mass of protein (in mg or μg) and (2) the absorbance of light at 595nm (the greater the amount of protein, the darker the solution and hence the more light absorbed as it passes through the cuvette). Since you will determine one of these parameters (absorbance at 595nm, or A595) for your isolated cell fractions, you can use the standard curve to determine the other parameter (μg of protein). You will be using bovine serum albumin (BSA, 1μg/μl) as a standard for the protein assay. We will run the samples in the plate reader, using sample plates. Your instructor will demonstrate. Run all protein assay samples (except blank) in duplicate (i.e. set up two identical wells). IMPORTANT: Immediately before taking an aliquot of any of your samples for use in the protein assay, be sure the contents of the sample are uniformly suspended. This is particularly important for your whole cell sample which has been sitting on ice for several hours so all the cells will have settled to the bottom of the tube. The best way to be sure the contents are evenly suspended is to flick the tube with your finger; ask the instructor how to do this. 1. Prepare dilutions of the BSA standard in microcentrifuge tubes as follows: Tube 1 (2/10 dilution): 20 µl BSA and 80 µl dH20 Tube 2 (4/10 dilution): 40 µl BSA and 60 µl dH20 Tube 3 (6/10 dilution): 60 µl BSA and 40 µl dH20 Tube 4 (8/10 dilution): 80 µl BSA and 20 µl dH20 2 2. Label your plate and plan the layout for your samples: Well dH2O(μl) BSA(μl) 1(blank) 10 -2 -10 (of 2/10 dilution) 3 -10 (of 4/10 dilution) 4 -10 (of 6/10 dilution) 5 -10 (of 8/10 dilution) 6 -10 (of undiluted) WC (whole cells) --CB(cell bodies) 5 -F(flagella) 5 -- sample(μl) ------10 5 5 3. AFTER adding all the solutions in the table THEN add 200 µl of BioRad protein reagent to each of these tubes. Allow tubes to sit at room temperature for at least 5 minutes (but no more than one hour), then read their absorbance at 595nm. Note: the readings for all your unknown samples must be within the range of your standards. If they are not, see your instructor immediately for further instructions. Record your data in Table I. 4. Mix 100 μl of each of your isolated Chlamydomonas fractions (WC, CB, and F, NOT your protein assay samples) with an equal volume (100 μl) of 2X (double strength) SDS PAGE sample buffer in a labeled microfuge tube. Be sure to designate your lab group as well as which sample is in the tube. The samples may now be placed in the freezer until the next laboratory period. 5. BEFORE next week's discussion, plot a standard curve, putting absorbance (A595) on the ordinate and μg protein (BSA) on the abscissa. Using your standard curve, determine the protein concentration of each of your three samples. Decide what volume of each protein sample to load on a gel, given the following information: a. You have mixed your sample with an equal volume of 2X (double strength) sample buffer before loading it on the gel. b. You will be using "mini-gels”, which can be run quickly and with minimal protein loads. You should (ideally) load 5-25 μl of sample (after it has been mixed with sample buffer) containing 2-50 μg of protein. If you do not have enough protein in some or all of your samples, then you should try to load the maximum volume of your least concentrated sample, and load close to the same mass of protein for your other samples, if that is feasible. Check your calculations with the lab instructor prior to loading samples onto the gel next week. c. You will be running duplicate lanes on your gel for each sample, so the maximum volume you can load in any one lane is half the total sample volume. 6. We will go over the use of the standard curve and the calculations for determining gel loads in discussion, however you should attempt to do these prior to coming to next week’s discussion. You may work on this with your lab partners or individually. Reference: Witman, G.B. 1986. Isolation of Chlamydomonas flagella and flagellar axonemes. Methods Enzymol.134:280-290. The assignment (due in discussion one week from lab): Flow chart (5 pt) Use the lab instructions (and if desired the reference above) to generate a flow chart of the purification procedure. For each step, indicate which fractions should contain a lot of, a little of, or no, flagellar proteins. Standard curve and volumes for sample loading (5 pt) See step 4 on p.3 in Protein assay section above. 3