Purification of Myosin Light Chain Kinase

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 μmleupeptin* - 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℃

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-PO4buffer 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℃,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℃).

4.Spin the homogenate in the Beckman J6 for 20 min at 4,000 RPM (4℃).Filter the supernatant through glass wool and discard the pellet (or save for light chain prep).Save 1 ml of supernatant (4℃)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℃).

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℃)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℃).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℃).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℃)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℃).

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℃),slowly add μltrapure (NH4)2SO4 (over15-30 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℃)(JA10 rotor).

13.Redissolve the pellets in a small volume of Buffer D (<50 ml).To this solution add the following (per50 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℃).

Volume of pellet solution ml.

14.Dialyze the above for 1 hr against 4 liters of Buffer D (4℃ - 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℃) and discard the pellet.Save 0.5 ml of supernatant for assay (4℃).

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℃].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℃).

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℃),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 60ml/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 10mM 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!