Myosin Light Chain Preparation

To be done the day before:

1.Put meat grinder and accessories in the cold room.

2.Put 6 one-liter centrifuge bottles in the cold room.

3.Put 14 to 16 500 ml Beckman polycarb centrifuge bottles with lids and inserts in the cold room.

4.Clean the 8 liter and the 28 liter beakers.

5.Put ETOH in the cold room.

6.Refrigerate or equilibrate the DE52 to be used.

7.Find parts to and assemble the 5½ cm ID column.

8.Make sure there is plenty of cold water in the cold room.

9.Thaw 8 M urea.

Solutions

Buffer A.Homogenizing Buffer: 28 liters: 50 mM KCl,20 mM Tris (pH 8.0),15 mM MCE,

0.2 mM MgAc2 (350 ml 4 M KCl,280 ml 2 M Tris,28 ml 15 M MCE,1.20 g MgAc2)

Buffer B.Triton X-100 Buffer: 4 liters of Buffer A plus 40 ml X-100 (1% Triton final)

Buffer C.Urea Homogenizing Buffer:

2.4 liters: 8 M urea,1 mM EDTA,15 mM MCE,20 mM

Tris (2.4 liters 8 M urea,12 ml 200 mM EDTA,2.4 ml 15 M MCE,24 ml 2 M Tris)

Buffer D.ETOH/MCE: 3 liters: For skeletal Muscle- 95% ETOH,15 mM MCE (3 liters ETOH,3 ml 15 M MCE)

For Cardiac Muscle- (1.5 liters ETOH 95%

(1.5 liters H2O,3 ml 15 M MCE)

Buffer E.Column Buffer: 16 liters:

20 mM Tris,15 mM MCE

(160 ml 2 M Tris,16 ml 15 M MCE)

Buffer F.Elution Buffer: 4 liters: 0.150 M KCl,10 mM Tris,15 mM MCE(150 ml 4 M KCl,40 ml 2 M Tris,4 ml 15 M MCE)

Buffer G.Dialyzing Solution: 8 liters:

4 mM MOPS (pH 7.0),15 mM MCE

(16 ml 2 M MOPS,8 ml 15 M MCE)

MCE = 2-mercaptoethanol (keep in cold room or refrigerator).

Day 1

1.Is cleaned of all fat and connective tissue,then sliced into strips and run through the meat grinder.Approximately 750 grams of tissue is needed.

2.Tissue is weighed (900 g).

3.The tissue is homogenized in 6 volumes of Buffer A using a large blender and blending for a total time of 90 seconds.It is best to bland in 30 second intervals with 30 second rests until total blend time is 90 seconds.

4.The homogenate is spun at 4200 rpm for 15 minutes in the Beckman J-6 using 1 liter bottles.

5.Discard the supernatant and place the pellets on ice.Add homogenizing Buffer A until the bottles are ½ full.Using the J-K homogenizer,resuspend the pellets 30 seconds.

Fill the bottles with Buffer A and centrifuge at 4200 rpm for 15 minutes in the Beckman J-6.

6.Repeat step 5 with Buffer B (A+ 1% Triton).

7.Repeat step 5 four more times with Buffer A until the pellet is nearly white and the supernatant is nearly clear (pellets may be frozen if rest of prep is performed later).

Discard supernatant and any loose white solid material which may be found on the compacted white pellets.