P. Fajerpage 1 of 9

Light Chain prep

Stock Solutions

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

Method

(All Steps Carried Out On Ice)

Washing myosin

  1. Take volume V of glycerol mixed myosin from the –20 oC freezer, and precipitate this myosin in V/2 * 14(approx.) volume of cold dH2O
  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 dH2O
  5. Centrifuge as before (1000*g for 20 min). Keep the pellet and discard the supernatant.

Removal of RLC

  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 dH2O 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.

Removal of TNB

  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.

Clarifying myosin

Centrifuge at top speed for 30 mins. Save the supernatant.

Dissociation of ELC from myosin

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 / 0.3 M
GdHCl / 5 M
EDTA / 5 mM
Tris-HCl / 45 mM
DTT / 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

Precipitation of myosin

  1. Add an equal volume of cold dH2O 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.

Removal of ethanol & guanidine

  1. Remove ethanol from the supernatant by rotary evaporation in 100-150 mls batches till EtOH stops flowing. Keep the bath temperature below 30 oC. Avoid solution bumping.
  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.

Concentrating ELC

  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 –70oC freezer.

Chromatography

  1. Might want to chromatograph on Cibacron Blue, Q-Sepharose or Mono-Q.

ELC preparation calculation sheet

Took ______ml of ______mg/ml of myosin in glycerol

(____mg/ml)(_____ml) = ______mg of myosin in glycerol

Wash w/ ______ml of dH2O

  1. Centrifuge, estimate pellet:______ml
  1. Dissolve pellet in: ______. Round off total ml of pellet by washing w/ dH2O
  2. Precipitate w/ 14* cold dH2O
  3. Centrifuge (discard supernatant), round off and estimate ml of pellet
  1. Dissolve pellet in:
  1. In glass cylinder add:
  2. incubate on ice for 10 minutes
  1. Precipitate w/14* vol of 0.002 M EDTA
    _____ml * 14= ______ml dH2O
    spin on stir plate for 10 minutes in cold box
  2. Centrifuge (discard supernatant)
  1. Dissolve pellet in:
  2. In glass cylinder:

a) incubate on ice for 10 minutes

b) Precipitate w/14* vol of 0.002 M EDTA
_____ml * 14= ______ml dH2O
spin on stir plate for 10 minutes in cold box

c) Centrifuge (discard supernatant)

  1. Dissolve pellet in:
  2. incubate on ice\cold box for 20 minutes
  1. Precipitate w/14* vol of 0.002 M EDTA
    _____ml * 14= ______ml dH2O
    spin on stir plate for 10 minutes in cold box
  2. Centrifuge (discard pellet)
  3. Dissolve pellet in:
  4. Centrifuge (KEEP SUPERNATANT)
  5. Store supernatant in guanidine solution overnight:
  1. 9.0M * ______L (soln) * 95.53g/mol = ______g of guanidine-HCl

DRY ICE

  1. Add equal volume of cold dH2O to denatured myosin sample. Add 66% cold ethanol
    ______%EtOH * x = ______(vol sample) * 3 – ______(vol sample)
    x= ______ml EtOH
  2. stir in cold box for 30 minutes
  3. Centrifuge ( KEEP SUPERNATANT)
  4. Remove EtOH by rotary evaporation
  1. Dialyze extensively in 0.1M DTT and %mM phosphate buffer:
  2. Determine concentration of ELC and concentrate (speed vac)

Light Chain separation on Cibacron Blue

Solutions

Precipitation Buffer:
(500 ml) / Mops pH = 7.0 / 10 / mM
EDTA / 2 / mM
Extraction Buffer×2:
(30 ml) / Tris pH = 8 / 80 / mM
guanidine·HCl / 10 / M
DDT / 5 / mM
EDTA / 10 / mM
Attention: guanidine·HCl does not dissolve completely!
Buffer A:
(4 l) / Tris pH = 7.5 / 20 / mM
DDT / 1 / mM
Buffer B:
(200 ml) / Tris pH = 7.5 / 20 / mM
DDT / 1 / mM
KCl / 1.2 / M

Myosin

For this prep purified myosin stored in 50% glycerol @ -20ºC 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 (720mg protein)and add 500 ml precipitation buffer
  • Precipitate the myosin for 20minutes
  • Spin the myosin down the myosin at 10k×g for 20 minutes and discard the supernatant

Extracting the Light Chains

  • Add 30ml of extraction buffer×2 to the myosin pellet and adjust the total volume with water to 60ml to have a protein concentration of 12mg/ml (The guanidine·HCl as well as the myosin dissolve)
  • Stir over night at 4ºC.
  • Dilute the mixture with 60ml cold water
  • Slowly add 240ml cold absolute ethanol
  • Stir for 30minutes at 4ºC 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 30ºC

Protect the oil pump using a cold trap filled with liquid nitrogen

  • Remove the guanidine·HCl by dialyzing 2× against 1l of bufferA (recycled from washing the cibacron blue column).

Leave some space in the dialysis bag because the volume will increase by about 30-50%

Preparing the column

Attention: Do this well ahead of time!

  • Pack 50ml 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 2.5×11cm
  • Wash the column was exhaustively with bufferA (approx. 0.5ml/min for 72h)

Have 4l of bufferA and flow them through the column over the weekend at an eluant hydrostatic pressure of about 20cm

Separation of the Light chains

  • Roughly estimate the amount of light chains assuming an UV absorbance at 280nm of 0.4 mg-1×cm-1
  • Apply light chains onto column (column works well for at least 60mg)
  • 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 1ml/min
Time / % Buffer B
0’ / 0% B
60’ / 0% B
240’ / 100% B
300’ / 100% B
  • Wash out LC3 and separate LC1 and LC2 by a linear gradient of 0–1.2M KCl at 1ml/min
  • Due to the long time this column runs use the EasyAG program at a sampling rate of 0.1Hz

Yield

The yields are calculated from UV absorbance assuming a distribution of LC2:LC1:LC3 of 3:2:1:

LC1 (20.7 kD): / 20mg (52%)
LC2 (19 kD): / 20mg (38%)
LC3 (16.5 (kD): / 14mg (92%)

Fajerlab under EDITMethods.doc10/20/1999 4:55 PM10/20/1999 4:55 PM