Leang, Ching 1-3
Membrane Fraction Preparation
1. Sign up for various centrifuges.
2. Make the following buffers:
a. 500 ml of 10 mM TrisHCl pH 8.0
i. 5 ml of 1M Tris/HCl pH 8.0
ii. ddH2O to 500 ml
b. 25% w/v Sucrose in 10 mM HCl pH 8.0
i. 25 g sucrose
ii. 75 ml 10 mM TrisHCI pH 8.0
iii. 20 mg/ml SodiumEDTA
iv. 1.37 ml 0.5 M EDTA pH 8.0
v. dH2O to 100 ml
c. 50 mM Tris/HCl pH7.5 + 5% glycerol
i. 0.83 ml 3M Tris.HCl pH7.5
ii. 5ml 50% glycerol
iii. dH2O to 50 ml
d. 6.4 mg/ml lysozyme
i. Make the day of the membrane prep
ii. 0.32 g lysozyme
iii. 5 ml 10 mM Tris HCl pH 8.0
iv. Put on ice
e. 0.1M MgCl2 + 10 mM TrisHCl pH 8.0
i. Aliquot 10 ml into 15 ml tube
ii. Add 100 ul of 1 M TrisHCl pH 8.0
iii. Store in the fridge
f. 5% (w/v) Brijj58
i. Chemical stored on shelf under P for polyoxyethylene cetyl ether
ii. Add 5 g Brijj58 to 100 ml dH2O in a beaker
iii. Stir all day.
iv. Will be a white cloudy solution.
3. Put all buffers on ice.
4. Gather four stir plates.
a. There are three in the glove bag.
5. Put centrifuge bottles on ice.
6. Pre-cool SS34 rotor
7. Label centrifuge bottles and caps.
a. You will have two bottles for each cell type.
8. Weigh one empty centrifuge bottle with cap per cell type
9. Mark bottle to note weight
10. Record weight on table.
11. Spin down 1 L cultures at 5000 x g for 20 minutes.
12. Remove supernatant
13. Gently resuspend cells from each centrifuge bottle in 100 ml of 10mM tris-HCl
pH 8.0.
a. Be fast or they’ll lyse.
Leang, Ching 2-3
14. Combine all resuspended cells of each cell type into weighed bottles.
15. Spin down cells at 5000 x g for 20 minutes.
16. Remove supernatant.
17. Get cells as dry as possible with pipetman.
18. Weigh bottles with cells to estimate wet weight.
19. Spin down cells at 5000 x g for 20 minutes.
20. Resuspend cells in appropriate volume of ice cold 25% sucrose, 10 mM Tris/HCl
buffer.
a. See table for volume
b. You do not have to be gentle.
21. Measure volume and record in table.
22. Transfer cells to beaker with stir bar and start stirring.
23. Add appropriate volume of lysozyme solution.
a. See table for volume
24. Incubate 15 min at RT while stirring.
25. During incubation:
a. Calculate total volume and enter in table
i. new vol1 = ml cell suspension + ml lysate
b. Calculate the volume of 20 mg/ml EDTA to add
i. See table for volume
26. Add calculated volume of 20 mg/ml EDTA
27. Incubate 15 min at RT while stirring.
28. Do these calculations while incubating:
a. Calculate total volume and enter in table
i. new vol2 = now vol1 + ml EDTA
b. Calculate appropriate vol. of 5% Brijj58 to add
i. See table for volume
c. Calculate total volume after Brihh58 addition and enter in table
i. new vol3 = new vol2 + ml Brijj58
d. Calculate the amount of DNAase to add and weigh out.
i. Accuracy is not important.
e. Calculate volume of MgCl2 to add
i. See table for volume
29. When 15 minutes are up add appropriate of Brijj58
30. Begin timer for incubation at RT while stirring.
31. As quickly as possible add DNAase and MgCl2.
32. Wash sticky DNAase powder off weigh boat with 1ml dH2O.
33. Complete 15 minutes at RT while stirring.
34. At end of incubation, set beaker on ice.
35. Transfer lysates to oakridge tubes.
36. Spin 15 minutes at 1,460 x g in SS34 at 4oC
37. Remove supernatant
a. Transfer to TI70 tubes.
b. Leave slimy phase behind
38. Balance tubes to 0.01 g
39. Spin 50000 x g for 1.5 hr in ultracentrifuge.
Leang, Ching 3-3
40. Rinse pellets with 5 ml 50mM TrisHCl + 5% glycerol
41. Repeat rinse a second time.
42. Resuspend pellets in volume recommended by Maddy.
43. Transfer resuspended pellets to microfuge tubes.