肌球蛋白轻链激酶的纯化
2010-05-26 22:59:29   来源:不详   评论:0 点击:

Solutions 1. 1 M Acetic Acid 2. Buffer A Homogenization 10 mM MOPS - 40 ml of 2M stock solution8 liters 4 mM EDTA - 1
Solutions
1. 1 M Acetic Acid
2. Buffer A Homogenization 10 mM MOPS - 40 ml of 2M stock solution
8 liters 4 mM EDTA - 160ml of 0.2M stock solution
1 mM DTT* - 8ml of 1M stock solution
0.1 mM PMSF* - 14ml of 10mg/ml stock solution
10 μg/ml trypsin inhibitor* - 8ml of 1mg/ml stock solution
pH 7.0
3. Buffer B DE 52 Wash 10 mM MOPS - 20ml of 2M stock solution
4 liters 1 mM EDTA - 20ml of 0.2M stock solution
1 mM DTT* - 4ml of 1M stock solution
0.1 mM PMSF* - 7ml of 10mg/ml stock solution
pH 7.0
4. Buffer C DE 52 Elution 10 mM MOPS - 5ml of 2M stock solution
1 liter 1 mM EDTA - 5ml of 0.2M stock solution
200 mM MaCl - 11.7g
1 mM DTT* - 1ml of 1M stock solution
0.1 mM PMSF* - 1.75ml of 10mg/ml stock solution
pH 7.0
5. Buffer D Dialysis 10 mM MOPS - 40ml of 2M stock solution
8 liters 0.5 mM EDTA - 20ml of 0.2M stock solution
1 mM DTT* - 8ml of 1M stock solution
0.1 mM PMSF* - 14ml of 10mg/ml stock solution
pH 7.0
6. Buffer E ø - sepharose 10 mM MPOS - 5ml of 2M stock solution
1 liter 250 mM NaCl - 17.55g
(Conductivity adjusted to 15m MHO)
0.5 mM EDTA - 2.5ml of 0.2M stock solution
1 mM DTT* - 1ml of 1M stock solution
0.1 mM PMSF* - 1.75ml of 10mg/ml stock solution
7. Buffer F CalM-Seph Equil. 10 mM MOPS - 5ml of 2M stock solution
1 liter 1 mM CaCl2 - 1ml of 1M stock solution
4 mM Mg (oAc)2 - 4ml of 1M stock solution
1 mM DTT* - 1ml of 1M stock solution
pH 7.0
8. Buffer G CalM-Seph Wash 10 mM MOPS - 10ml of 2M stock solution
2 liters 1 mM CaCl2 - 2ml of 1M stock solution
4 mM Mg (oAc)2 - 8ml of 1M stock solution
200 mM NaCl - 23.4g
1 mM DTT* - 308mg
10 μM Leupeptin* - 2ml of 5mg/ml stock solution
0.1 mM PMSF* - 3.6ml of 10mg/ml stock solution
1 μg/ml Pepstatin A* - 2ml of 1mg/ml stock solution
pH 7.0
9. PMSF 500 mg
10mg/ml 50 ml 2 propanol
store in dark bottle @ 25°C
10. Leupeptin 35 mg
5 mg/ml 7 ml dd H2O
Store frozen @ -60°F
11. Pepstatin 2.5 mg
1 mg/ml 2.5 ml D MSO
Store frozen @ -60°F
12. DE-52 300 ml
Equilibrate in Buffer B
pH 7.0
Conductivity < 3 milli MHO
13. Tripsin
inhibitor
8 mg
8 ml H2O
store frozen -20°F
14. DTT 4.63 g
1 M 30 ml H2)
store frozen -20°F
15. TLCK 320 mg
20 mg/ml 16 ml H2O
pH < 6.0 with acetic acid
16. TPCK 180 mg
20 mg/ml 9 ml EtOH
17. K-PO4
buffer
0.3M KH2PO4 10.21g in 250 mL H2O
~0.5 L 0.3M K2HPO4 10.21g in 250 mL H2O
titrate the K2HPO4 into the KH2PO4 until pH at 6.8
18. Recycle CaM-sepharose by running through several columns volume of 1M Kcl in Buffer F. Store
on Buffer F with 0.2% NaN3.
PROCEDURE
All prep is done at 4°C, except procedure number 1.
1. Two rabbits should be injected (ear vein) with ~5 cc of nembutal solution and then exsanguinated and
skinned. Skeletal muscle should be cleaned of fat and connective tissue and kept on ice.
2. The tissue should be ground in the cold room using the fine setting of the meat grinder and weighed.
Should get a 1 kg tissue from 2 rabbits.
Weight of tissue: G.
3. In a Waring blender, homogenize with 6 volumes of Buffer A mixing for 30 sec., then rest 20 sec.; total
of 3 times (4°C).
4. Spin the homogenate in the Beckman J6 for 20 min at 4,000 RPM (4°C). Filter the supernatant
through glass wool and discard the pellet (or save for light chain prep). Save 1 ml of supernatant
(4°C) for assay. Add pepstatin and TLCK to final concentrations of 1 μg/ml and 40 Og/ml,
respectively.
Volume of supernatant #1 Ml.
5. The pH should be adjusted to 5.7 then more slowly to 5.5 with 1 M HoAc. Stir 5 min to stabliize pH.
Spin the solution in the J6 for 30 min at 4,000 RPM (4°C).
6. Filter the supernatant #2 through the glass wool and adjust the pH to 6.7 with solid NaHCO3. Save
1 ml of supernatant #2 (4°C) for assay.
Volume of supernatant #2 Ml.
7. Adjust the conductivity downward to 2.6 m MHO with cold Buffer B.
8. DE52 (150 g, dry = 300 ml, wet) previously equilibrated in Buffer B (can equilibrate without DTT and
PMSF) should be added. Adjust pH to 6.8. Resultant slurry stirred for 60 min (4°C). Collect the resin
in a 2,000 ml scintered0glass funnel (saving the unbound pool) and wash with 4 liters of Buffer B
*using water vacuum and frequent gentle stirring to avoid compacting the resin (4°C). Save 1 ml of
the unbound pool.
Volume of unbound pool ml.
9. Add the washed resin to 150 ml of Buffer C and increase the conductivity carefully to 12 mMHO with
solid NaCl. Stir the slurry for 10 min (4°C) and collect resin on 2,000 ml scintered-glass funnel. Wash
the resin with 200-300 ml of additional Buffer C. Save 1 ml of this pool for assay (4°C).
Volume of DE52 eluate pool ml.
10. To each portion of Buffer C used add the following (per liter):
1.75 ml of PMSF stock (10 mg/ml)
0.1 ml Leupeptin stock (5 mg/ml)
1.0 ml Pepstatin stock (1 mg/ml)
2 ml TLCK (20 mg/ml)
3 ml TPCK (20 mg/ml)
11. While stirring the DE52, eluate pool slowly (no frothing - (4°C), slowly add ultrapure (NH4)2SO4 (over
15-30 min) to a final saturation of 55% (333 g/liter). After all solubilized, stir for an additional 15 min.
(NH4)2SO4 added G.
12. Spin the solution in the JC-21 for a5 min at 8,000 RPM (4°C) (JA10 rotor).
13. Redissolve the pellets in a small volume of Buffer D (<50 ml). To this solution add the following (per
50 ml):
90 μl of PMSF stock (10 mg/ml)
50 μl of Pepstatin stock (1 mg/ml)
50 μl of Leupeptin stock (5 mg/ml)
Save 0.5 ml for assay (4°C).
Volume of pellet solution ml.
14. Dialyze the above for 1 hr against 4 liters of Buffer D (4°C - spectraphor 12-14 K MW cutoff). Repeat,
total 8 liters/2 hr. Check conductivity of dialyzed pellet solution; stop dialysis when at 15 mMHO.
Volume of diluted solution ml.
Conductivity mho.
15. Spin the sample at 50,000 RPM in the 50.2 Ti rotor and Beckman Ultracentrifuge for 30 min (4°C)
and discard the pellet. Save 0.5 ml of supernatant for assay (4°C).
Volume of high spin pool ml.
16. Apply the supernatant to a phenyl-sepharose column pre-equilibrated with Buffer E. Collect the flow
through in 15 ml fractions. Pool peak I. Wash column with Buffer E without CaCl, Peak II.
17. Add the following to Peak I (per 100 ml):
100 μl Pepstatin stock (1 mg/ml)
100 μl Leupeptin stock (5 mg/ml)
200 μl CaCl2 stock (1 M)
400 μl Mg (oAc)2 stock (1 M)
Mix well - do not froth. Save 0.5 ml for assay.
Volume of concentrate ml.~10 mMHO
18. Adjust conductivity to ~10 mMHO with Buffer F. Load the above solution on to a calmoudlinsepharose
column (20 ml Bed volume pre-equilibrated with Buffer F; max. 0.6 mg kinase/ml CaMsepharose).
Collect unbound pool in 250 ml beaker (pump = 20x). When completely loaded, gently
layer ~5 ml of Buffer C on top of the resin, connect the column top and wash overnight at 45 ml/hr with
Buffer G [Abs = 1.0, chart speed 0.5 mm/min, pump = 35x, 6.6 min/tube (5 ml fractions), 4°C]. Under
these conditions in the morning, recorder pen should be back down to baseline. Save 0.5 ml of the
two pools for assay (4°C).
Volume of unbound pool ml.
Volume of wash pool ml.
19. To 190 ml of Buffer G add 10 ml of the 200 mM EGTA stock = 10 mM EGTA. Remove wash buffer
from top of resin and carefully layer on 5 ml of the EGTA-Buffer G solution. Reconnect the column top
and elute at 45 ml/hr. A280, chart speed 1 mm/min, Abs = 0.5, (4°C), 2 racks (A), drop = 45 (~2 ml
fractions), or pump = 35x, 2.6 min/tube.
20. Pool the A280 peak, saving 0.5 ml for Bradford, assay, and SDS gel. Measure volume and freeze the
pool in 10% glycerol at -60°F.
Volume of CaM-Sepharose pool ml.
21. Run a 7.5% SDS gel to assess purity (may need further purification on hydroxylapatite). Measure
enzyme activity.
22. The CaM-Sepharose pool is dialyzed for ~ 4 hr against 10 mM KPO buffer (pH 6.8), 1 mM DTT and
3 mM NaN3. 1 L changed 2 times (3L total dialysis).
23. A 10 ml hydroxylapatite column (Bio-Rad, Bio-Gel HTP; 1.5 x 6 cm) was equlibrated in buffer: 10 mM
potassium, pH 6.8, 1 mM DTT, and 3 mM NaN3. Collect fractions of ~ 1 ml/tube. Flow rate is 60
ml/hr. After application of protein pool to column, the column is washed for 1.5 hr with same buffer -
no detectable protein should wash from the column.
24. The protein is eluted overnight from the HA column with a 400 ml H2KPO4 (pH 6.8) gradient, from 10
mM to 300 mM KPO4, with 1 mM DTT.
25. Samples are analyzed by SDS-PAGE, mini gel.
26. Two pools of protein are collected: Pool I (main peak I) and pool II (trailing edge and bump). Add
glycerol to ~ 10% of pool volume and store at -60°.
27. Good work. Have a beer!
 

相关热词搜索:肌球 球蛋 蛋白 白轻 轻链 链激 激酶

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