Revised 4/91, SGH
Check water in cold room, need at least 28 liters for actin acetone powder, 32 liters for CapZ prep. (based on 1kg muscle from 4 breasts)
Put meat grinder and stirrers in cold room.
Put 8 liters Acetone in cold room.
Check supply of cheesecloth. Order Grade 40 mesh (24x20 threads/inch from Fisher). Be careful if you borrow some that it is the correct mesh size!
Will need ultracentrifuge and Ti-45 rotor on Day2 if making actin.
Check stocks of protease inhibitors for Day2. Will need:
All quantities are based on starting with 1kg of muscle.
1) Make 4 liters of Myosin extracting solution with cold water. Do not need to pH this solution. Make before getting breasts, or day before prep.
2) Use (4) fresh chicken breasts from the local abattoir, put directly into ice when received, and do the rest of the procedure in the cold room.
3) Dissect away pectoralis major and minor, being careful to leave behind skin and fat.
4) Grind muscle two times through meat grinder and weigh using student balance. After last piece of meat is through, put ice in grinder to remove more meat. Make gel sample. Minimize time between grinding and extracting solution.
5) Divide meat into two 4 liter beakers, each with 2 liters of cold myosin extracting solution. Stir each with an overhead stirrer for 15 min. Hug method uses two overhead stirrers. We have one motor with paddle and one home-made paddle that works; Carl Frieden has another overhead motor that can be borrowed.
6) Add 2 liters (less if doesn't fit) of cold dd H2O to each. Stir 5 min.
7) Strain through 4 layers of cheesecloth using large funnel. (Do not skimp on size as the piece never seems to be big enough, I like 3ft x 3ft. It is OK to reuse the same piece)
8) Suspend each 1/2 of meat in 3 liters (less if necessary) cold dd H2O. Stir 5 min. Strain through cheesecloth as above.
9) Suspend each 1/2 of meat in 3 liters of cold 0.4% NaHCO3 (need 6l total of this--12 g/3l). Stir 15 min. Strain through cheesecloth as above.
10) Repeat #9
11) Pour 2 liters cold dd H2O over each 1/2 of muscle in funnel, and strain.
12) Resuspend each 1/2 in 2 liters of cold acetone. Do this and the following procedures using acetone in the fume hood. Stir 5 min. Strain through cheesecloth. Put waste acetone into sink diluted with lots of water.
13) Repeat #12. Strain well to remove as much acetone as possible, speeds drying.
14) Air dry meat over a large sheet of filter paper, in fume hood, with blower on for maximum airflow over meat. If convenient, can "stir up" muscle after a couple hours to speed up drying. Make gel sample of extracted muscle. Weigh acetone powder.
15) If not starting CapZ prep immediately, store dry acetone powder in seal-a-meal bag at -70°C. Should produce about 100 g CSM acetone powder.
12 hours total (if you're efficient)
Buffer G. Need 40 ml/g acetone powder.
1 liter 4 liters
1. Buffer G 50X stock in freezer. 20 ml 80 ml
(100 mM Tris/HCl pH 8.0, 10 mM
ATP, 5 mM DTT, 0.25% NaN3)
2. CaCl2, 1 M. 0.2 ml 0.8 ml
3. PMSF, 1 M in DMSO 0.1 ml 0.4 ml
4. Pepstatin A, 1 mM in abs EtOH 0.1 ml 0.4 ml
5. Leupeptin, 1 mM 0.1 ml 0.4 ml
6. Benzamidine, 0.1 M 1 ml 4 ml
1 M KCl Buffer. Need 45 ml/g acetone powder.
1 liter 4 liters
1. KCl, dry 75 g 298 g
2. EDTA, 0.5 M 0.5 ml 0.8 ml
3. PMSF, 1 M in DMSO 0.1 ml 0.4 ml
4. Pepstatin A, 1 mM in abs EtOH 0.1 ml 0.4 ml
5. Leupeptin, 1 mM 0.1 ml 0.4 ml
6. Benzamidine, 0.1 M 1 ml 4 ml
0.6 M KI Buffer. Need 30 ml/g acetone powder.
1 liter 4 liters1. KI, dry 99 g 398 g
2. Na thiosulfate, dry 3.16 g 12.64 g
3. beta-mercaptoethanol, liquid 0.4 ml 1.6 ml
4. NaN3, 10% 1 ml 4 ml
5. Tris/HCl, pH 7.2, 1 M 10 ml 40 ml
6. PMSF, 1 M in DMSO 0.1 ml 0.4 ml
7. Pepstatin A, 1 mM in abs EtOH 0.1 ml 0.4 ml
8. Leupeptin, 1 mM 0.1 ml 0.4 ml
9. Benzamidine, 0.1 M 1 ml 4 ml
10. EDTA, 0.5M 0.5 ml 0.8 ml
Buffer B with 50 mM KCl. Need 8 l for dialysis.
1 liter 8 liters
1. KCl, dry 3.73 g 29.82 g
2. beta-mercaptoethanol, liquid 78 µl 624 µl
3. NaN3, 10% 1 ml 8 ml
4. Tris/HCl, pH 8.0, 1 M 10 ml 80 ml
5. PMSF, 1 M in DMSO 0.1 ml 0.8 ml
6. EDTA, 0.2 M 0.5 ml 4 ml
1. Mix acetone powder with cold Buffer G, 20 ml/g acetone powder (2 liters), for 30 min. Stir in cold room with overhead paddle stirrer.
2. Separate soluble extract from muscle residue by filtering through 4 layers of cheesecloth as in acetone powder prep. Save first Buffer G extract for actin prep, otherwise discard.
3. Repeat Buffer G extract (2 liters).
4. Extract muscle residue with 1 M KCl Buffer, 15 ml/g (1.5 liters), 60 min.
5. Repeat 1 M KCl extract (1.5 liters), except stir for only 30 min.
6. Repeat step 5.
7. Extract muscle residue with 0.6 M KI Buffer, 15 ml/g (1.5 liters), 30 min. Save extract.
8. Repeat KI extract, except use 7.5 ml/g (0.75 liters). Save extract.
9. Repeat step 8. Save extract. Make gel samples of extracts and leftover CSM
10. Combine KI extracts, dialyze against 8 l of water. Use the largest size dialysis tubing, which must be checked for pinholes, and the plastic pipet holder.
Use medium size funnel to fill tubing.
Do not overfill bags, as they will expand considerably with dialysis. (leave >10cm)
After about 4 hrs, change dialysis solution to 8 liters of Buffer B with 50 mM KCl. (easiest to add a more concentrated stock of Buffer B and add then add ddH2O to the level with original 8 liters water. (Usually around 11 liter mark.)
11. Wash DEAE Cellulose column
a) Remove cellulose from column into 4 liter beaker. Add dd H2O to 4 liters. Add some (~50g?) additional new DE-53 resin in case there are losses during washing procedures.
b) Add 165ml 12 N HCl. Stir 15-20 minutes.
c) Wash on Büchner funnel with 8 liters dd H2O. These washes can be with house distilled water, not necessary to use the MilliQ-water. (Use 2 pieces Whatman #541 filter paper, and make sure no cellulose is lost. Use Whatman 541 or else the paper will disintigrate!!!)
d) Return to beaker, add ddH2O to 4 liters. Add 165 ml 12 NaOH, or 104 ml 19 N NaOH (50%) Stir 15-20 minutes.
e) Wash with 8 liters dd H2O as before.
f) Return to beaker, add dd H2O to 4 liters, 40 ml 1 M Tris pH 8.0, 4 ml 10% NaN3
g) pH to 8.0 with HCl. Otherwise nothing will bind.
It is easier at this point to make up 2 liters of a 4x stock of Buffer B without KCl, then use portions at 100 mM KCl for packing and rinsing the column and 100mM and 400mM KCl for the gradient.
12. Bring up 1/3 of the beads in about 1.5 liters of Buffer B with 100 mM KCl. Use these to pack the bottom 1/3 of the column either today, or tomorrow while spinning KI extracts.
Do not let the column run dry.
Keep remaining 2/3 of beads in the cold room.
13. Sign up for a Sorvall centrifuge for tomorrow AM.
Time is variable, depending on how fast the column flows during sample loading. To avoid a late night, get the sample loading as soon as possible.
1. Dialyzed KI extract should have white ppt. Spin in GSA rotor in Sorvall at 13,000 rpm for 15 min. Save a sample of the supernatant. Measure volume, A280, and make an SDS gel sample.
2. In a 4 liter beaker, mix spun sample with the leftover 2/3 of the beads.
Mix with a stirrer bar in the cold room, making sure that any clumps of beads are broken up.
3. Apply sample to DEAE column using loading chamber. The column will not hold all the sample at once, so mix the sample and beads before adding more.
Use high pressure (ceiling to floor) so that gradient can be started overnight.
Collect the flow through and make a gel sample. The flow through may contain significant unbound CapZ?? If it does consider reloading flow through after first DEAE run.
4. Prepare gradient. 2 liters of Buffer B with 100 mM KCl and 2 liters of Buffer B with 400 mM KCl. (1.8 liters of each fits better, and allows for stirring in 2 liter glass beakers). Add 0.1 mM PMSF to both. Beakers should be on top shelf, on four spot stirrer plate.
5. Start gradient immediately when sample has finished entering column. Do not wash with any column buffer.
6. Start collecting fractions when gradient starts. Fractions 350 drops (nearly fills 16 mm tubes, but can decrease to 300 if tubes are overfilling). One fraction collector full (116 tubes) is enough to collect the CapZ, which elutes early. The remainder can go to waste.
7. UA5 settings: Abs range = 1, Chart speed = 0.6 cm/hr.
8. Equilibrate S-200 column for tomorrow with Buffer B with 0.1 mM PMSF.
9. Dialyze G150 actin for falling ball viscosity assays. 5 mg for 50 assays should be enough for DEAE and S-200. Weigh out 15 mg or G150 actin into 5 ml G-buffer and dialyze against G-buffer.
Time: about 6 hrs. Can do both days 4 and 5 here by 1) starting early, 2) dialyzing the AmSO4 P40-75 for only one hour, and 3) running the S-200 column at maximal flow rate (ceiling to floor pressure).
1. Analyze DEAE fractions by FBV. CapZ activity should appear in first absorbance rise after return to baseline following sample flow thru. 5 µl of fraction should lower viscosity to near that of water.
2. Pool best 20-25 fractions of peak activity (no more) and measure volume and A280.
3. (Optional) Measure conductivity of fractions adjacent to pool and that of Buffer B with both 100 and 400 mM KCl to calculate salt concentration in CapZ pool. Should be about 180 mM KCl. Make gel sample from pool.
4. Am2SO4 - 40% cut. Add 243 g solid Am2SO4 per liter of CapZ pool. Stir on ice in cold room for 30 min (15-20 if in a hurry). Spin in GSA rotor in Sorvall, 13,000 rpm for 15 min. Discard pellet. Measure volume of sup and save it.
(313 g/liter if doing old style 50-75 cut)
5. Am2SO4 - 75% cut. Add 245 g solid Am2SO4 per liter of Sup50 (This is not the same as the volume of the previous pool).
(176g/liter if doing old style 50-75 cut)
Stir on ice and spin as in previous step. Discard sup. Save pellet and suspend P40-75, in minimal volume (10 ml) of Buffer B and dialyze against Buffer B for 1-2 hrs.
6. Spin resuspended, dialyzed 40-75 pellet in SA600 rotor for 10 min. Sterile filter the sup, measure volume and A280. Make gel sample.
7. Apply sample to S-200 column. Start collecting fractions immediately. UA5 settings: 150 drops/tube, Abs range = 0.5, chart speed 0.6 cm/hr.
About 4 hrs.
1. Analyze fractions by FBV.
2. Pool 5-6 fractions of the peak centered around fraction 28. Measure volume and A280 and make gel sample.
3. Dialyze pool against 10 mM MES, pH 6.0, 0.1 mM PMSF overnight.
pH at room temp, dialyze in cold room.
NOTE: Consider this modification: Dialyze S200 pool against 10 mM MES, pH 6.5, 0.1 mM PMSF, overnight to remove salt, and then switch to same buffer at pH 6.0 for 30-60 min just before running the MonoS column. This may prevent the loss of capZ by precipitation at the lower pH.
4. Prepare solutions for MonoS column. Need 500 ml each. Sterile filter.
A: 10 mM MES, pH 6 prepare using cold water (standardize pH meter with RT stds)
B: A with 1 M KCl. prepare using cold water (by mixing MES and KCl, then adjusting pH with KOH.
5. Get the MonoS column ready to run, i.e., set up column on FPLC, wash with buffer A to get rid of ethanol and apply saw-tooth wash and equilibrate in buffer A. This requires that the FPLC buffers are prepared in advance (see preparation of buffers below).
8 hrs, including running of gel.
1. Remove sample from dialysis, sterile filter. If there is alot of ppt. material, spin it out before filtration.
2. Turn on FPLC, wash A and B into system. Rinse Superloop and column (16x10 MonoS from Frieden/Gordon, stored in Gordon lab.) with A. Column flow at 2 ml/min. Consider a salt gradient to wash column if it has been used for another application. Turn on UV lamp.
3. In manual mode, apply sample to column with Superloop at 2 ml/min. Run chart recorder at 0.4 cm/min. Abs range = Total # mg in sample ÷ 10. (Usually 0.5). Wash column with 5 ml of A.
4. Set fraction collector for 0.5 min. Mode to 0, time (button 7) to 0.5.
5. Run gradient program, called Method File #9. Check the program by listing the method. The steps should be:
0.0 CONC %B 0.0
0.0 ML/MIN 2.0
0.0 CM/MIN 0.40
0.0 PORT.SET 6.1
45.0 CONC %B 13.5
46.0 CONC %B 100 DO NOT RUN GRADIENT
46.0 PORT.SET 6.0 THROUGH SUPERLOOP
66.0 CONC %B 100
67.0 CONC %B 0
80.0 ML/MIN 0.0
80.0 CM/MIN 0.0
Turn off UV lamp when done with gradient
5. Pour at least 3 10% SDS gels while the column is running. Run peak fractions from the column on the gel. Do every fraction across the peak and check the fringes of the peak.
6. Pool fractions, based on gel appearance. Measure A280. Calculate yield of each pool: Ext. Coef. at 280nm is 1.25 ml/mg.cm.
7. Any CapZ that will not be used within 2 weeks (max) should be frozen in glycerol.
8. If CapZ is to be used soon, dialyze into 5 mM Tris pH 8.0, 0.1 mM DTT, 1 mM EDTA, 0.02% NaN3, 0.1 mM PMSF. Sterile filter into Siliconized tubes for storage at 4°C.
