Add 2mL of buffer III to each centrifuge tube (after remainder of supernatant has been discarded). Swirl gently and pour out solution in sink. (This constitutes one "wash" of the ribosomal pellet).
Place tubes in ice bucket and add 4.0mL of buffer III to each tube.
Using a clean glass rod with a rounded fire-polished end, dislodge the pellet and break it up into small chunks. Squeeze the gelatinous chunks into small pieces with frequent working of glass rod. It is best to trap pieces against side of tube and then "roll" rod across the pieces in order to flatten them out. After all gelatinous chunks are gone, ribosomes will be "resuspended", and solution will be "pearly opalescent".
Transfer ribosomes solution to one clean SS-34 tube. Balance with blank tube.
Spin, 15K, 15 min, 4°C, SS-34 to pellet crud.
Add 20mL of buffer IV to each of ten 50.2 Ti tubes.
Layer supernatant from SS-34 spin on top of buffer IV solution in 50.2 Ti tubes using a Pasteur pipet. (Discard pellet in SS-34 tube). Balance 50.2 Ti tubes.
Spin, 4°C, 50.2 Ti overnight (put ultracentrifuge on "hold"). Set speed at 22K if run will be 12 hours long; 20K for 14 hours; 19K for 16 hours; 24K for 10 hours.
DAY 2:
Discard supernatant in each tube. Wash each pellet with 2mL of buffer III (as in step 66).
Repeat steps 67 through 70.
Add 18mL of buffer IV to each of eight 50.2 Ti tubes.
Layer supernatant from SS-34 spin on top of buffer IV solution on 50.2 Ti tubes (about 6.0mL per tube) using a pasteur pipet. Balance 50.2 Ti tubes. (Discard pellet in SS-34 tube).
Spin, 50K, 150 min, 4°C, 50.2 Ti.
Discard supernatant in each tube. Wash each pellet with 2mL of buffer III (cf. step 66).
Repeat steps 67 through 70.
Add 20mL of buffer IV to each of six 50.2 Ti tubes.
Layer supernatant from SS-34 spin on top of buffer IV solution i 50.2 Ti tubes using a pasteur pipet. Balance 50.2 Ti tubes. (Discard pellet in SS-34 tube).
Spin as in step 73.
DAY 3:
Discard supernatant in each tube. Wash each pellet with 2mL of buffer V.
Add 2mL of buffer V to each tube. Resuspend pellets as before.
Turn on 37°C water bath. Turn on absorbance spectrophotometer.
After pellets have been resuspended, pool ribosomes in a 25mL graduated cylinder. Mix gently. Place on ice.
Remove 1uL of ribosome solution, place in assay tube, add 999uL of buffer V, and vortex. Measure A260 and A280, using buffer V as blank and reference. The A260 of the stock ribosome solution will be 1000X the measured A260.
Add enough buffer V to the ribosome solution to make its A260 equal to 300. (For example, if A260 of ribosome stock solution=900 in step 87, and you have 8mL of the solution, add 16mL of buffer V to the ribosomes. Mix ribosomes gently.
Add a volume of 0.1M puromycin to ribosomes which is equal to 1/100 of total volume of ribosomes. (That is, if total volume of ribosomes in step 88 is 24mL of 0.1M puromycin). Mix solution gently but thoroughly.
Incubate ribosomes at 37°C for 60 minutes.
Test a piece of medium diameter dialysis tubing for leaks (fill neat top with ddH2O, then put gentle pressure on tubing using nitrogen tank, with 10mL pipet stuck in tygon tubing of tank). Then pour water out of tubing.
At end of incubation, transfer ribosomes to dialysis tubing.
Place ribosomes/dialysis tubing in 4 litres of buffer VI. Note time.
Total dialysis time should be 22-24 hours, and there should be tow changes in dialysate. Ideally, ribosomes should be moved from one 4 litre beaker to another at 8 hours and at 16 hours. But arrange times so no one has to come in late at night, making sure that the minimum time per change is 5 hours. (e.g., put on dialysis at 1 p.m., change at 6-10 p.m., and change at 8 a.m.) Check to see if someone is working in the lab that night and could change it for you if you wouldn't otherwise be in. If everything proceeds as planned, this dialysis should end about noon on Day 4.
DAY 4:
Transfer dialyzed ribosomes to a 50mL graduated cylinder on ice. Calculate the number of 50.2 Ti tubes needed in the next step (the maximum volume of ribosomes to be added to one 50.2 Ti tube will be 8mL in step 97).
Add buffer VII to 50.2 Ti tubes as needed to obtain full tubes. For example, for 36mL of ribosomes, you would add 18mL of buffer VII to 4 tubes and 22mL of buffer VII to 2 tubes (one tube will be used as a blank for balance); then you would add 8 mL of ribosomes to 4 tubes, 4mL to one tube, and 4mL of H2O to the blank. Be sure to keep track of which tube is the blank, and note the number of the hole in the rotor in which the blank is placed.
Layer ribosomes solution on top of buffer VII in 50.2 Ti tubes using pasteur pipet. Balance.
Spin, 50K. 150 min., 4°C, 50.2 Ti.
Discard supernatant in each tube. Wash each pellet with 2mL of buffer I (as in step 66).
Place tubes in ice and add 4.0mL of buffer I to each tube. Break up pellets with glass rod as in step 68, and let the ribosomes resuspend overnight. Cover the tubes tightly with parafilm.
DAY 5:
Repeat steps 68-70.
Add 20 mL of buffer VII to as many 50.2 Ti tubes as you spun in step 99.
Layer supernatant from SS-34 spin on top of buffer VII in 50.2Ti tubes using pasteur pipet. Balance. (Discard pellet in SS-34 tube).
Spin, 50K, 150 min. 50.2 Ti, 4°C.
Discard supernatant in each tube. Wash each pellet with 2mL of buffer VII.
Add 2mL of buffer VII to each tube.
Repeat steps 68 through 70.
Label 0.5mL microfuge tubes with "RH" (for Ribosomes, High-salt-washed) and the page number of the prep (e.g., "ET-197"). You may also want to color-code the top of the tubes.
Transfer supernatant from SS-34 spin into clean graduated cylinder. (Discard pellet in SS-34 tube).
Record volume of ribosome solution.
Remove two 2uL aliquots for absorbance readings. Add 998uL of buffer VII to each assay tube and vortex. Turn on absorbance spectrophotometer.
Using a pipetman or a plastic pipet and pipet bulb, place a 50 or 100 ul aliquot of RH into each labeled microfuge tube.
Hold RH storage tubes vertically and quick-freeze them in liquid nitrogen. Place tubes in 75°C freezer for storage. Mark their location on the inventory sheets.
Measure and record A260 and A280 of diluted ribosome aliquots (from step 112).
Assay ribosomes for polyphe activity, for poly(rU)- and EF-Tu-dependent binding of Phe-tRNA, and for integrity of rRNA (by sedimentation in a SDS-sucrose gradient).
PREP SOLUTIONS
Buffer I
Final Concentration
For 2 litres, add:
10.0mL of 4.0M NH4Cl
20mM
20.0mL of 1.0M Tris-HCl (pH 7.6)
10mM
20.0mL of 1.0M MgCl2
10mM
1.0mL of undiluted beta-EtSH (2-mercaptoethanol) ddH2O to
1 litre
7mM
Buffer II
For 1 litre, add:
50.0mL of 4.0M NH4Cl
200mM
10.0mL of 1.0M Tris-HCl (pH7.6)
10mM
10.0mL of 1.0M MgCl2
10mM
0.5mL of undiluted beta-EtSH ddH2O to 1 litre
7mM
Buffer III:
For 1 litre, add:
250mL of 4.0M NH4Cl
1000mM
10.0mL of 1.0M MgCl2
10mM
10.0mL of 1.0M Tris-HCl (pH 7.6)
10mM
0.5ml of undiluted beta-EtSH ddH2O to 90-950mL. Titrate back to pH 7.6 with 4 N KOH ddH2O to 1 litre
7mM
Buffer IV
For 1 litre, add:
250mL of 4.0M NH4Cl
1000mM
10.0mL of 1.0M MgCl2
10mM
10.0mL of 1.0M Tris-HCl (pH 7.6)
10mM
0.5mL of undiluted beta-EtSH
7mM
85.6 grams of sucrose. Add water to 900-950mL after sucrose dissolves, titrate
back to pH 7.6 with 4 N KOH ddH2O to 1 litre.
250mm
Buffer V:
Prepare this solution in a graduated cylinder just before use.
For 200mL, add
10.0mL of 1.0M Tris-HCl (pH 7.4)
50 mM
20.0mL of 4.0M KCl
400 mM
10.0mL of 4.0M NH4Cl
200 mM
8.0mL of 1.0M MgCl2
40 mM
0.1mL
of undiluted beta-EtSH ddH2O to 200mL
7 mM
Buffer VI:
For 4 litres, add:
214 grams of NH4Cl
1000 mM
40.0mL of 1.0M Tris-HCl (pH 7.4)
10 mM
40.0mL of 1.0M MgCl2
10 mM
2.0mL fo undiluted beta-EtSH ddH2O to 4 litres
7 mM
Buffer VII:
For 1 litre, add:
5.0mL of 4.0M NH4Cl
20 mM
10.0mL of 1.0M MgCl2
10 mM
10.0mL of 1.0M Tris-HCl (pH 7.6)
10 mM
0.5mL of undiluted- EtSH
7 mM
85.6 grams of sucrose. Add water to ~900mL after sucrose dissolves, add ddH2O
to 1 litre.
