page 1 of 9 P. Fajer
3 M KCl
0.2 M potassium or sodium phosphate buffer, pH 7.0
0.2 M Tris-HCl buffer, pH 8.6
0.5 M EDTA
0.5 M DTNB
0.5 M DTT
(All Steps Carried Out On Ice)
1. Take volume V of glycerol mixed myosin from the –20 o C freezer, and precipitate this myosin in V/2 *
14(approx.) volume of cold dH
2
O
2. Centrifuge using 250 ml Nalgene bottles at 1000*g for 20 minutes. Use the white plastic round plates that go at the bottom of the tube holders in the rotor to prevent the tubes from breaking.
3. Dissolve the pellet with KCl and sodium or potassium phosphate buffer, pH 7.0 to make the final concentration of 0.5 M KCl and 5 mM phosphate buffer. Then add DTT to make the final concentration 1 mM DTT.
4. Precipitate with 14* volume of cold dH
2
O
5. Centrifuge as before (1000*g for 20 min). Keep the pellet and discard the supernatant.
1. Dissolve the pellet with KCl and Tris-HCl buffer to get a final concentration of 0.5 M KCl and 20 mM Tris-HCl buffer.
2. Check the concentration of myosin. The optimum concentration is 10-15 mg/ml. Save for SDS.
3. Add 10 mM EDTA and 10 mM DTNB. Incubate on ice for 10 minutes. Solution should turn a yellowish reddish color.
4. Precipitate with 14* volume of cold dH
2
O and make the final concentration 2 mM EDTA
5. Centrifuge the filaments as before. Keep the pellet.
6. Dissolve in 0.5 M KCl, 20 mM Tris-HCl.
7. Repeat the procedure if neccessary.
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P. Fajer
1. Dissolve as in 0.5 M KCl and add 5mM DTT. Incubate in DTT for 20 minutes.
2. Precipitate with water.
3. Centrifuge the filaments. Keep the pellet.
4. Dissolve in 0.6 M KCl, 10 mM Tris-HCl buffer, and add 5 mM DTT. page 2 of 9
Centrifuge at top speed for 30 mins. Save the supernatant.
Mix 1:1 volumes of 8.5-9.0 M guanidine-HCl (make sure it is dissolved) in 10 mM EDTA, 5 mM DTT, 80 mM Tris-
HCl solution and myosin at 20 mg/ml. Alternatively, dissolve the myosin pellet in the stock solution of GdHCl and then bring to correct concentration.
The final concentrations are:
Final conc.
Myosin 10 mgs/ml
KCl
GdHCl
EDTA
Tris-HCl
DTT
0.3 M
5 M
5 mM
45 mM
2 mM
PH 8.0
Keep the sample in the following solution on a stir plate overnight (or longer) at room temperature.
DRY ICE IS NEEDED FOR 2nd DAY OF PREP
1. Add an equal volume of cold dH
2
O to the myosin sample.
2. Add cold ethanol (EtOH) to 66% slowly to form precipitate of the denatured heavy chain.
3. Stir gently for 30 minutes in the cold box.
4. Centrifuge at 1000xg for 20’. Keep the supernatant. Remove floaties (floating denatured myosin). The pellet is gelatinous denatured heavy chain.
5. If necessary, spin again.
1. Remove ethanol from the supernatant by rotary evaporation in 100-150 mls batches till EtOH stops flowing.
Keep the bath temperature below 30 o C. Avoid solution bumping .
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P. Fajer page 3 of 9
2. Dialyze extensively against 4 volumes of 1 mM DTT and 5 mM sodium phosphate buffer, pH 7. Change buffer twice.
3. Keep any precipitate (LC2/LC1 mixture) for future chromatographic separation in urea.
1. Determine concentration of ELC: Abs(280-320).
2. Concentrate to 1.5-2 mg/ml or lyophilizeand store in 1.8 ml vials in –70 o C freezer.
1. Might want to chromatograph on Cibacron Blue, Q-Sepharose or Mono-Q.
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P. Fajer
Took _______ ml of ______ mg/ml of myosin in glycerol
(____mg/ml)(_____ml) = _______mg of myosin in glycerol
Wash w/ ______ml of dH
2
O
1. Centrifuge, estimate pellet:_______ml
M
 x

0 .
005 M NaP0
4
; x

____ ml of _ M NaP0
4 ml
M
 x

0 .
001 M DTT ; x

____ ml of _ M DTT ml
M
 x

0 .
5 M .
KCl ;.........
.
x

____ ml .
of ml
_ M KC l
2. Dissolve pellet in: _______ . Round off total ml of pellet by washing w/ dH
2
O
3. Precipitate w/ 14* cold dH
2
O
4. Centrifuge (discard supernatant), round off and estimate ml of pellet
M
 x

0 .
5 M .
KCl ; x

____ ml of ml
_ M KC l
5. Dissolve pellet in:
M
 x

0 .
01 M EDTA ; x

____ ml of ml
_ M EDTA
M
 x

0 .
01 M DTNB ; x

____ ml of ml
_ M DTNB
M
 x

0 .
01 M EDTA ; x

____ ml of ml
_ M EDTA
6. In glass cylinder add:
7. incubate on ice for 10 minutes
M
 x ml dH
2
O

0 .
002 M EDTA ; x

____ ml of _ M EDTA
8. Precipitate w/14* vol of 0.002 M EDTA
_____ml * 14= _______ml dH
2
O
spin on stir plate for 10 minutes in cold box
9. Centrifuge (discard supernatant)
M
 x

0 .
02 M TRIS HCl ; x

____ ml of ml
_ M Tris HCl page 4 of 9
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P. Fajer
M
 x

0 .
5 M KCl ; x

____ ml of ml
_ M KCl
10. Dissolve pellet in:
11. In glass cylinder:
M
 x

0 .
01 M DTNB ; x

____ ml of ml
_ M DTNB
M
 x

0 .
01 M EDTA ; x

____ ml of ml
_ M EDTA page 5 of 9 a) incubate on ice for 10 minutes
M
 x ml dH
2
O

0 .
002 M EDTA ; x

____ ml of _ M EDTA b) Precipitate w/14* vol of 0.002 M EDTA
_____ml * 14= _______ml dH
2
O
spin on stir plate for 10 minutes in cold box
13. c) Centrifuge (discard supernatant)
M
 x

0 .
02 M TRIS HCl ; x

____ ml of ml
_ M Tris HCl
14. Dissolve pellet in:
15. incubate on ice\cold box for 20 minutes
M
 x

0 .
005 M DTT ; x

____ ml of _ M DTT ml
M
 x

0 .
6 M KCl ; x

____ ml of ml
_ M KCl
M
 x ml dH
2
O

0 .
002 M EDTA ; x

____ ml of _ M EDTA
M
 x

0 .
005 M DTT ; x

____ ml of _ M DTT ml
M
 x

0 .
5 M KCl ; x

____ ml of ml
_ M KCl
19.
20.
21.
Precipitate w/14* vol of 0.002 M EDTA
_____ml * 14= _______ml dH
2
O
spin on stir plate for 10 minutes in cold box
Centrifuge (discard pellet)
Dissolve pellet in:
22. Centrifuge ( KEEP SUPERNATANT )
23. Store supernatant in guanidine solution overnight:
M
 x

0 .
005 M DTT ; x

____ ml of _ M DTT ml
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P. Fajer
M
 x

0 .
08 M TRIS HCl ; x

____ ml of ml
_ M Tris HCl
M
 x

0 .
01 M EDTA ; x

____ ml of ml
_ M EDTA
24. 9.0M * _______L (soln) * 95.53g/mol = __________g of guanidine-HCl page 6 of 9
DRY ICE
25. Add equal volume of cold dH
2
O to denatured myosin sample. Add 66% cold ethanol
_______%EtOH * x = ________(vol sample) * 3 – ________(vol sample) x= ______ml EtOH
26.
27. stir in cold box for 30 minutes
Centrifuge ( KEEP SUPERNATANT)
28. Remove EtOH by rotary evaporation
M
 x

0 .
005 M NaP0
4
; x

____ ml of _ M NaP0
4 ml
M
 x

0 .
0001 M DTT ; x

____ ml of _ M DTT ml
29. Dialyze extensively in 0.1M DTT and %mM phosphate buffer:
30. Determine concentration of ELC and concentrate (speed vac)
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P. Fajer
page 7 of 9
Precipitation Buffer:
(500 ml)
Extraction Buffer × 2:
(30 ml)
Mops pH = 7.0
EDTA
Tris pH = 8 guanidine·HCl
DDT
EDTA
Attention: guanidine·HCl does not dissolve completely!
Buffer A:
(4 l) Tris pH = 7.5
DDT
Buffer B:
(200 ml) Tris pH = 7.5
DDT
KCl
10 mM
2 mM
80 mM
10 M
5 mM
10 mM
20 mM
1 mM
20 mM
1 mM
1.2 M
For this prep purified myosin stored in 50% glycerol @ -20 ºC was used. Fresh prepared myosin or crude extract from myofibrils or frozen meat should work as well but were not tried so far.

Take 60 ml of myosin in storage buffer (720 mg protein)and add 500 ml precipitation buffer

Precipitate the myosin for 20 minutes

Spin the myosin down the myosin at 10k × g for 20 minutes and discard the supernatant

Add 30 ml of extraction buffer × 2 to the myosin pellet and adjust the total volume with water to 60 ml to have a protein concentration of 12 mg/ml (The guanidine·HCl as well as the myosin dissolve)

Stir over night at 4 ºC.

Dilute the mixture with 60 ml cold water
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P. Fajer page 8 of 9

Slowly add 240 ml cold absolute ethanol

Stir for 30 minutes at 4 ºC to precipitate the heavy chains

Spin the heavy chains down at 12k × g for 20 minutes

Remove the ethanol by rotary evaporation

Control the temperature not to exceed 30 ºC

Protect the oil pump using a cold trap filled with liquid nitrogen
 Remove the guanidine·HCl by dialyzing 2 × against 1l of buffer A (recycled from washing the cibacron blue column).

Leave some space in the dialysis bag because the volume will increase by about 30-50%
Attention: Do this well ahead of time!

Pack 50 ml Cibacron Blue 3GA (Type 3000-CL, Sigma-# C-1535, Lot 93H9512, 5.0
 mol Cibacron Blue 3GA per ml of gel) in a column to give a gel bed of 2.5 × 11 cm

Wash the column was exhaustively with buffer A (approx. 0.5 ml/min for 72h)

Have 4 l of buffer A and flow them through the column over the weekend at an eluant hydrostatic pressure of about 20 cm

Roughly estimate the amount of light chains assuming an UV absorbance at 280 nm of 0.4 mg -1 × cm -1

Apply light chains onto column (column works well for at least 60 mg)

LC3 is eluted in the void volume whereas LC1 and LC2 bind to the column

Start the following program on the gradient mixer after all the light chain mix has sunken into the gel
Programming the gradient mixer at 1 ml/min
Time
0’
% Buffer B
0% B
60’
240’
0% B
100% B
300’ 100% B

Wash out LC3 and separate LC1 and LC2 by a linear gradient of 0 –1.2 M KCl at 1 ml/min

Due to the long time this column runs use the EasyAG program at a sampling rate of 0.1 Hz
The yields are calculated from UV absorbance assuming a distribution of LC2:LC1:LC3 of 3:2:1:
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P. Fajer
LC1 (20.7 kD):
LC2 (19 kD):
20 mg (52%)
20 mg (38%)
LC3 (16.5 (kD): 14 mg (92%)
Separation of Light Chains on Cibacron Blue
0.14
LC2
0.12
LC1
0.10
2.5
2.0
1.5
0.08
0.06
Myosin Peak 1 Peak 2 Peak 3 Peak 2 Myosin
1.0
0.04
0.5
LC3
0.02
0.00
0 20 40 60 80 100 120 140
Time [min]
160 180 200 220 240 260 280
0.0
page 9 of 9
Abs.
cond.
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