Pyrene Actin Prep

from notes of Chris, in actin notebook (20 Feb 91) and Jane (14 Dec 87).

 -Starting material is lyophilized conventional actin (2/3rds sucrose by weight) made from  chicken skeletal muscle acetone powder.   Fresh (unlyophilized) conventional actin may also be used.

-Dissolve actin in buffer G with no DTT at approximately 3 mg/ml.  Start with 100-300 mg of actin (300-900 mg of lyophilized actin with sucrose).

Buffer G: 0.2 mM ATP, 0.2 mM CaCl₂, 2 mM Tris/HCl, pH 8.0 @ 25°,  +/-  0.5 mM DTT

                                Stock      1 l       2 l        4 l
2 mM Tris/HCl, pH 8 @ 25°     1 M        2 ml     4 ml       8 ml
0.2 mM ATP                  0.1 M        2 ml     4 ml       8 ml
0.2 mM CaCl₂                  1 M      0.2 ml   0.4 ml     0.8 ml
0.5 mM DTT                    dry       77 mg   154 mg     308 mg 

-Dialyze at 4 degrees with several changes of buffer until well dissolved; leave a small air bubble in tubing and use this to mix contents twice a day by inverting the tubing.  This will break up chunks of actin and speed dialysis.

 -Remove from dialysis tubing and determine concentration by A₂₉₀ vs dialysate: 

e = 0.63 ml/(mg-cm);      = 2.66 x 10⁴   /(M-cm)

 -Dilute to 1 mg/ml with dialysis buffer.  Polymerize by making 100 mM KCl and 2 mM MgCl₂, and stirring slowly at room temperature for 20 minutes.

 -Dissolve pyrene (mw 385) in DMF (10 mg/ml); vortex to dissolve.  Add to actin at  10:1 molar ratio pyrene: actin (ca 10 mg pyrene/ 100 mg actin).  Add  while stirring, and continue stirring at 4 degrees in the dark (cover beaker with foil).  A white, cloudy precipitate may form.  Continue stirring overnight.

 -Dialyze vs  buffer G with 0.5 mM DTT in smallest diameter tubing practical with several changes until actin is in solution.  A white precipitate may form in tubing. 

 -Consider removing excess pyrene precipitate with low-speed centrifugation.

 -Polymerize by making 100 mM KCl and 2 mM MgCl₂, and stirring slowly at room temperature for 20 minutes. 

 -Centrifuge in Ti 45 rotor at 4 degrees, 40,000 rpm for  1 hour. 

 

-Resuspend yellow and brown pellet in buffer G with DTT to approximately 5 mg/ml.  Homogenize with loose fitting dounce plunger.  Dialyze at 4 degrees against the same buffer with several changes.

 -Centrifuge in swinging bucket rotor (Sw 41 Ti) 25,000 rpm for 2 hours.  Carefully remove top 2/3 rds of supernatant with pipet, avoiding  mixing with lower 1/3 rd and pellet.   Transfer to new tube.  Gel-filter on foil-wrapped G-150 column equilibrated with buffer G with DTT.  Cover fraction collector with foil or run in dark. Collect 4 ml fractions.  Pool fractions from top and back of second peak, as for G-150 actin prep.  Determine concentration and pecent labelling by A₂₉₀ and A₃₄₄.   Since pyrene absorbs at 290 nm, must correct the actin absorbance at this wavelength.

 for pyrene:  e at A₃₄₄ = 2.22x10⁴ / (M-cm)

for actin:  corrected A₂₉₀ for actin X* = X - 0.127Y

                                                                     where X = A₂₉₀, Y = A₃₄₄

 

% labelled = [pyrene]/[actin]; expect 60-70 %.

 Should recover 30-40 % of starting actin.

Options for Storage:

-Add 2 grams  sucrose per gram actin, freeze in lyophilizer flask with liquid nitrogen, and lyophilize.  Cover with foil.  Transfer to scintillation vials and store at -20 in the dark. 

-Consider aliquoting to smaller tubes before freezing and lyophilizing; then just have to add buffer G and dialyze when using each aliquot.  ?5-10 mg actin/ tube.

- Just rapid freeze in G buffer. No sucrose or lyophilization.

 

 

N-(1-pyrene)iodoacetamide, from Molecular Probes, catalog number P-29.   Store at -20 C,  dessicated in dark.  mw 385.