Myosin Light Chain Preparation
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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.
Day 2
8.Remove about 1 liter of pellets and place in the blender.Using about 500 ml of Buffer C,rinse the bottles and add to the blender.Add 2 liters of Buffer C and approximately 50 g solid urea and homogenize for 2 minutes in 30 second intervals in a large blender.
If frozen pellets are used,thaw the pellets in the cold room over-night.Put the pellet in the blender the next morning and add 2 liters of Buffer C to blender and homogenize for 2 minutes.
9.Pour mixture (white and foamy)into an 8 liter beaker.Rinse the blender with remaining Buffer C and add to beaker.Stir at room temperature for 2 hours using large blade and drill (Craftsman)(set speed at 2; check the speed occasionally; sometimes it will go faster).
10.Add equal volume of Buffer D in the cold room and stir for 1 hour at 4℃.
11.Centrifuge at 8000 rpm for 20 minutes at 4℃ using the Beckman J21-C and 500 ml prechilled polycarb bottles with inner seals *usually 14 bottles- 3 spins are necessary;rotor JA10).
12.Filter the supernatant through glass wool into an 8 liter beaker saving supernatant.
Discard pellets and rinse bottles immediately with water.Soak bottles in the Beckman soap.
13.Add 800 ml of regenerated DE52 (if using dry preswelled DE52,measure out at least 350 g and equilibrate in Buffer E)to the filtered supernatant.Let this absorb overnight with stirring,at 4℃.
Day 3
14.Stop stirring and allow the DE52 to settle (½ hour to 1 hour).When a distinct line is visible between the DE%2 and the liquid,siphon off as much ETOH supernatant as possible.
15.Divide the DE52 into 1 liter bottles,fill with Column Buffer E and centrifuge in the J-6 for 10 minutes at 1000 rpm.
16.Discard supernatant and refill bottles with Column Buffer E.Shake slightly so that the DE52 is resuspended in the buffer.Spin in the J-6 for 10 minutes at 1000 rpm.
Repeat for a total of 4 washes.
17.Pack the DE52 into the 5½ cm ID column in the cold room.
18.When all of the DE52 has been put in the column and has packed somewhat,take a 10 sample of the eluting fluid to see if any protein is still being eluted (Coomassie
Assay- add dye to the protein sample and read against a sample of water and dye at A595 on the spectrophotometer).When it has been determined that no more protein is being eluted and the DE52 is packed,take the Buffer E off of the top of the column with a syringe.Then carefully layer the Elution Buffer F on top of the column to a height of about 2 inches.
19.Fill a 4 liter beaker with Elution Buffer F.Collect 20 ml fractions (250 drops per tube).
The adaptor top to the column may need to be used if the DE52 is packed too low in the column.This step will run overnight.Set the speed at about 1 drop/sec.
Day 4
20.A.Run Coomassie Blue Assay on every other tube.Take 10 ? samples,add dye,and read against a water blank at A595 on the spectrophotometer; graph A595 against fraction number.Absorbance should rise quickly,fall slightly,and then tail-off slowly.Pool tube under first sharp high protein peak (Pool 1)and pool remaining tubes under broad lower protein peak (Pool 2).Find volumes of
Pools 1 and 2.
B.Measure conductivity of every fifth tube.Allow the conductivity meter to sit in the cold room for 30 minutes before using it n the cold room; graph mMHO against the fraction number.
21.Put samples in lyophilizer bottles and freeze in liquid nitrogen.Put on lyophilizer and leave until dry (overnight or over weekend).
Day 5
22.Resuspend separate light chain pools 1 and 2 in a small volume of Dialyzing Solution G.Pipette into dialyzing tubing (size #20)through a small funnel.Tie three knots in
each end (leave some air in tubing so it will float)and put in 8 liters of Dialyzing Solution G.Change to a fresh solution of Dialyzing Solution G after a few hours to force a new equilibrium to occur.Dialyze overnight.
Day 6 and after
23.Centrifuge in J21℃using JA20 rotor at 15,000 rpm for 20 minutes.Take a small sample for gel and assays and freeze the remainder in -60℃ freezer (note volume).
24.Determine the protein concentrations of Pools 1 and 2 with the Coomassie Blue Assay and B.S.A.standards.Run 10% SDS gels.
25.Do 32P assay to determine maximal extent of phosphorylation of light chain.
26.Scan gels with spectrophotometer at A 600.Cut out and weigh the peaks to determine % impurities and percent of light chain 1 and light chain 2 (and light chain 3).Take a photograph of the gels.
