Ribosome(R), S-100 enzymes(S), and peptidyl-tRNA hydrolase(H) preps
(as of 7/31/89)
DAY -2:
Nuclease-free techniques should be used throughout. This is especially during the ribosome purification. Also, all samples should be kept in ice or at 4°C throughout.
Sign for ultracentrifuge and 50.2 Ti rotor; will use them from ~ 11 a.m. on Day 1 through the morning of Day 2; and at other times depending upon how ribosomes will be prepared.
Clean centrifuge tubes and bottles for GSA, 50.2 Ti, and SS-34 rotors.
Clean mortar (~17 cm O.D.) and pestle; set in cold room so that it will be cold when needed.
Sign for fraction collector; will use afternoon of Day 1. Also line up stirrers.
Make sure the Sephadex G-25 and DEAE-cellulose columns are recycled and re-equilibrated. If necessary, re-equilibrate them. If the DEAE-cellulose is new, it must be washed with high salt and then re-equilibrated back into low salt before use in prep.
Check stock on:
DNase I (Worthington DPFF or DPRF; RNase-free) (need ~600ug)
alumina (Sigma A305) (need ~250g)
dialysis tubing
liquid N2 ampules or microfuge tubes for storage
ammonium sulfate
puromycin (need ~ 25mg for RH prep)
phenylmethylsulfonyl fluoride (PMSF; need ~20uL of 0.01M)
DAY -1:
Plan to wear warm clothing tomorrow. Starting at 7:00-7:30 a.m. is advisable.
Weigh out ~100g of cells (no more than 150g) from frozen stock. Place into aluminum foil and store at -20°C overnight. Record lot number and mass of cells used.
Weigh out alumina (2.5 times the mass of cells used in step 2) into weighing boat, cover with Al foil, and place in cold room overnight.
Prepare solutions that will be needed. Some solutions will not be used, depending upon the specific ribosomal prep procedures being followed. Buffer I (a total of at least 5 L); Buffer VIII (at least 3L); Buffer IX (at least 4 L); 1L of 1.0M KOH; 1L of 1.0M Tris- HCl (pH 7.6)l etc.
Cool solutions overnight in cold room.
Be sure rotors are cold.
DNase I should be stored at 4°C at 1mg/mL concentration in 0.15M NaCl. Do not shake solution, since this denatures then enzyme.
DAY 1:
Break frozen cells into fairly small chunks using a hammer wrapped in aluminum foil (do not do this in the mortar, for it will break) so that cells will thaw faster. Then place frozen cells in mortar and let stand in cold room to thaw cells.
Begin grinding with pestle when cells are fairly well thawed. Add alumina in portions during grinding until the total mass of alumina added is 2.5 times the mass of cells.
When cell sap is at "popping" stage (i.e., when every pestle push leads to pooping), add buffer I to mortar in small aliquots, with mixing by pestle, (as one does in making cream of mushroom soup) in order to avoid clumping and loss of solution uniformity. The total volume (in mL) of buffer I added should be 2 times the number of grams of cells that were used.
Add DNase to mortar to final concentration of 3ug DNase/mL buffer I added. (0.6mL of 1mg/mL DNase stock solution should be added if 200mL of buffer I have been added).
Add PMSF to mortar to final concentration of 10uM. (200uL of 0.01M PMSF should be added if 200mL of buffer I have been added).
Let solution incubate in cold room with occasional stirring for a total of 30 min following DNase addition. If the DNase digestion has been completed, the solution should no longer be viscous, and the cell sap solution should drip freely from the pestle.
Pour solution into two GSA bottles. (Another bottle may be required if more than 100g of cells are ground up). Balance tubes.
Spin, 10K, 15 min, GSA, 4°C to remove alumina and unbroken cells.
Turn on ultracentrifuge in order to cool it to 2-4°C. Fill a couple of ice buckets with ice, and keep samples in (on) ice during the following steps.
Carefully pour supernatant from GSA spin into a clean 400mL beaker. Do the pouring in one smooth pour, without interruption. It is OK if some sediment goes into the beaker. [Discard residue (pellets) in GSA bottles in waste basket; do not wash down sink].
Pour the supernatant into 6 to 8 SS-34 tubes and balance tubes.
Spin, 15K, 15 min. 4°C, SS-34 to complete removal of alumina and unbroken cells.
Carefully pour each supernatant with one movement into a fresh 400mL beaker. (Discard pellets into waste basket).
Pour supernatant into 8 (or more, if necessary) 50.2 Ti tubes Balance.
Spin, 25K, 25 min., 50.2 Ti, 4°C to complete removal of cell debris.
Carefully pour supernatant into a fresh 400mL beaker. (Discard pellets).
Pour supernatant into fresh 50.2 Ti tubes. Balance tubes. Note: tubes spun at 50K must contain at least 22mL of solution to avoid tube collapse.
Spin, 50K, 120 min. (or, if tubes are showing too many stress lines, 40K, 4hrs), 50.2 Ti, 4°C to pellet ribosomes.
Clean centrifuge tubes and bottles, as well as mortar and pestle, with a brush to ensure that all debris is removed. Then soak all items (except the aluminum 50.2 Ti caps) in Micro soap and warm water. Wash with tap water and distilled water as usual, and bake glassware in oven. Do not allow 50.2 Ti O-rings to soak too long in soap. Wash rotors after use and let dry, but be sure to cool them so they are ready when needed. These clean-up procedures, as well as wiping down benches, in lab and cold room, should be done whenever there is free time in order to stay ahead of the dirty dishes.
After the step 18 spin, use a 10mL pipet and pipet bulb to carefully remove the top 70% of the supernatant form each tube and transfer to a clean 250mL beaker. Keep tip of pipet below surface of supernatant just far enough to avoid air bubbles going up the pipet. The capacity of this type of tube is 26mL, so 70% of a full tube would equal 18.2mL (Pour rest of supernatant out; SAVE PELLETS, which contain the ribosomes, and place tubes in ice. Go to "ribosomal pellet" section for instructions on how to proceed).
Pour supernatant in beaker into fresh 50.2 Ti tubes and balance.
Spin, 50K, 120 min., (or 40K, 4hrs), 50.2 Ti, 4°C to remove the rest of ribosomes.
Re-equilibrate Sephadex G-25(3.1cm ID x 22.5cm)[which gets rid of small molecules that bind DEAE and lower capacity] and DEAE-cellulose(5.8cm ID x 10.5cm)[which gets rid of tRNA], columns in buffer I (500-600mL though each G-25 column, and 800-1000mL through the DEAE column) in cold room at 4°C.
Carefully remove the top 70% of supernatant from each tube and transfer to a fresh 250mL beaker. (Discard remainder of supernatant, as well as pellets. Note: pellets have to be cleaned out of tubes using a brush.)
Load one-half of remaining yellow-colored supernatant (maximum of 60mL) onto Sephadex G-25 column which has been re-equilibrated in buffer I, and run column in cold room with buffer I as eluent. When yellow material nears bottom of column, begin fraction collector (Gilson, 150 drops/fraction; yields about 9mL/fraction). Collect yellow fractions, and then let rest of eluent run into waste container.
When the separation in step 25 is about half-done, load the other half of yellow supernatant onto the second G-25 column, and run it as in step 25.
Pour all yellow (look down into tube from the open end to estimate color intensity) fractions (about 16-18 total) directly from fraction tubes onto top of DEAE-cellulose column which has been re-equilibrated in buffer I at 4°C. Begin fraction collector (Gilson, 150 drops/faction) when the S-100 begins to wash into column. After the S-100 has loaded onto column, wash DEAE-cellulose column with buffer I.
Turn on absorbance spectrophotometer in order to warm up UV lamp.
Wash 300mL through each of the Sephadex G-25 columns after the yellow fractions have been collected. This will re-equilibrate columns for the next purification (step 35). CAUTION: do not forget columns and let them run dry.
After 60 fractions (~9mL each) have been collected from the DEAE-cellulose column, change elution buffer to buffer II (replace both the buffer above the DEAE cellulose resin and the buffer in the solvent reservoir). Going form 20-200 mM salt is sufficient to elute the protein fairly quickly but tRNA comes off slowly- it is the tRNA you are getting rid of.
Remove 60 fractions collected in buffer I and cover tubes with Handi-wrap. This eluent contains the peptidyl-tRNA hydrolase enzyme and will be referred to as the "hydrolase" fraction. (Go to "hydrolase" section to see how to proceed with this material).
Place a rack of clean tubes in the fraction collector. Start column and fraction collector, and collect 30-40 tubes.
Read the A280 and A260 of 1:10 dilutions of the yellow fractions eluted from the DEAE-cellulose column with buffer II (use buffer II in reference cuvette). Use at least 0.5mL per absorbance assay to get an accurate measurement.
Pool in a 250mL beaker those fractions with A280>5 and A280 /A260>1 (that is, fractions whose dilutions have A280 >0.5 and 280/260 > 1).
Load yellow fractions pooled in step 34 onto Sephadex G-25 column and run as before in buffer I (step 25). If total volume of those pooled fractions is more than 60mL, split the sample and run one-half over each of the two Sephadex G-25 columns.
Pool fractions with a strong yellow color (looking end-on) in a clean 250mL graduated cylinder. Mix gently, but thoroughly. This is the S-100 enzyme (="S") solution.
Record total volume of S solution.
Remove 250uL of S solution for use tomorrow. Place in glass assay tube and freeze at -20°C.
Label 5mL liquid N 2 ampules, as well as a few 2mL size ampules, with "S" and the page number of the prep (e.g., "ET-198"). Dispense S into these tubes, at 4mL per 5ml ampule and 1mL per 2 mL ampule. Hold tubes vertically and quick-freeze them in liquid nitrogen. Place tubes in -75°C freezer for storage. Mark their location on the inventory sheets.
DAY 2:
Dilute two 100uL aliquots of S (from -20°C sample of step 38 above) with 900uL of buffer I. Determine A260 and A280 of the S solution using buffer I as blank and reference.
Determine mg/ml of S solution by Lowry assay, using two 1uL and two 3uL aliquots of S. (Do Lowry of hydrolase solution [step 31 above] at the same time).
Wash 400mL of buffer VIII through each G-25 column. Label columns as containing buffer VIII.
Wash, in turn, the following through the DEAE-cellulose column
1 litre of 0.1M KOH
4 litres of H 2 O
1 litre of 1 M Tris-HCl (pH 7.6)
2 litres of buffer VIII
Resuspend resin in column during H 2 O wash, without disturbing the glass wool plug at the bottom of the column. Label column as containing buffer VIII.
PEPTIDYL-tRNA HYDROLASE (from step 31):
Day 1 (continued):
Read A280's of fraction in hydrolase eluent. Pool fractions that have an A280> 0.1 in a 400mL beaker. There may be two peaks of A280; if so, pool only the first peak. The total number of fractions in the first peak should be approximately the same as the number of fractions added to the DEAE-cellulose column initially. Note which fractions have been pooled.
Add stirring bar to beaker and stir gently in cold room. (Cover with Al foil).
Measure the volume of a fraction not pooled with the hydrolase solution and multiply this volume by the number of fractions that were pooled. This gives you the total volume (v, in mL) of the hydrolase solution.
Weigh out 0.56 V grams of ammonium sulfate into a weighing boat.
Add solid ammonium sulfate slowly, with stirring, to the hydrolase solution in the cold room. Avoid accumulating large amounts of solid ammonium sulfate on the bottom of the beaker.
After the ammonium sulfate has dissolved, stir the solution for 30 min at 4°C.
Pour solution into GSA centrifuge bottle(s). Balance.
Spin, 10K, 40 min, GSA, 4°C to pellet the precipitated protein.
Lift bottles out of rotor and place in ice carefully, because the pellets will break up easily if disturbed.
Aspirate supernatant from GSA bottle using a pipet connected to aspirator vacuum. (at end, suck water in ice bucket through pipet to wash out pipet and tubing).
Resuspend pellet(s) in 2mL of buffer IX.
Transfer hydrolase solution to small diameter dialysis tubing.
Dialyze against 2 litres of buffer IX at 4°C overnight.
Day 2:
Place hydrolase dialysis tubing in a fresh 2 litre batch of buffer IX and dialyze for at least 5 more hours.
Transfer hydrolase (including any crud in solution) to an SS-34 tube. Balance with blank.
Spin, 15K, 20 min, SS-34, 4°C to pellet crud.
Label two 2mL liquid N 2 (or 1.5mL microfuge)tubes with "H" and the page number of the prep (e.g., "ET-198").
Transfer supernatant into the storage tubes. (Discard pellet).
Measure and record total volume of H using Pipetman or plastic pipet and pipet bulb.
Transfer 90uL of H solution into an assay tube. Add 810uL of buffer IX and vortex gently. Measure A280 and A260. Save solution for activity assays. [If these are not to be done today, freeze in liquid N 2 ampule and label it "H diluted, assay tube, (page)"].
Remove two 3uL and two 5uL aliquots for Lowry assay ( see step 41).
Hold H storage tubes vertically and quick freeze them in liquid nitrogen. Place tubes in -75°C freezer storage. Mark their location on the inventory sheets.
DNase I (Worthington DPFF or DPRF; RNase-free) (need ~600ug)