P. Fajerpage 1 of 3
Acetone Powder
1)Kill rabbit by CO2 asphixiation, bleed, gut, skin & ice 20. Remove back and leg muscle (white only). Remove fat, facia and red meat with scissors. Cut into ~ 1 chunks.
2)In cold room. Grind muscle in meat grinder and weigh “rabbit burger.”
3)Add 3 volumes of Guba Straub solution (no EDTA) and extract 10 minutes. (Removes myosin.)
4)Dilute with equal volume of cold deionized water, and let settle 5 mins. So cheesecloth doesn’t get so clogged.
5)Squeeze through 2 layers of cheesecloth.
6)Add 5 volumes of cold deionized water to residue and mix 5 mins. Allow to settle 5 mins.
7)Squeeze through 2 layers of cheesecloth, discard the liquid.
8)Wash twice with 5 volumes of 0.4% NaHCO3* for 10 min wash and for 5 min. for second wash, with stirring (further washes are necessary with red muscle). Squeeze through 2 layers of cheesecloth after each washing.
9)Wash residue twice with 5 volumes of deionized water and squeeze through 2 layers of cheesecloth. Do these washings as quickly as possible.
10)Wash with 3 volumes of cold acetone (4-6C). Extract 10, stirring occasionally.
11)Repeat acetone washing until acetone remains clear on addition of an equal amount of water, 6-8 washings are usually required. Allow ~1L acetone (spectro grade)/wash.
12)Let resulting powder dry overnight at room temperature under a hood and between 2 layers of cheesecloth; put some sort of weights along the sides of the cheesecloth cover.
13)Place in tared 60ml Nalgene bottles and write weight of the powder on the bottle cover tightly and store in the walk in freezer.
Solutions
*0.4% NaHCO3 is wt/vol percent and pH 8.5 at 25C.
*GUBA-STRAUB SOLN.
.3 M KCl
.15 M K Phos pH 7.0
To make V
use.12 V of 2.5 M KCl
.10 V of 1 M K2HPO4
Acetone Powder Prep Sheet
Ref: Spudich and Watt (1971) J.B.C., 246, 4866-4871. ; Thomas et al (1979) J. Mol. Biol., 132, 257-273.
Set-up:
Ahead of Time:Order a large (8-12 lbs.) New Zealand White Rabbit (by noon Friday for the previous week), and make sure there is CO2 available (tank near the wetlab desk. Prepare the buffers needed for the prep.
In the Wetlab:Cover the benchtop with aluminum foil near the sink. Ready the rabbit box and 2 large green plastic bags, 1` plastic tray and 2 buckets of ice, clean scissors, tweezers, scalpels, and a supply of fresh scalpel blades.
In the Coldbox: Cover the table with aluminum foil. Have the meat grinder and accessories, a large funnel and ringstand, 18-20 double layers of cheesecloth, 4-5 large plastic weigh boats, 2 4L beakers, 2 2L beakers, and several smaller ones, a stopwatch, a supply of plastic transfer pipettes, and supply of small spatulas, and a large stir-rod and/or spatula chilled and ready.
In the Hood:Clear and cover an area with aluminum foil to dry the acetone powder over night in between two layers of cheesecloth. Store in a 500ml plastic bottle.
Solutions:
BufferpH Chemical[Stock]Volume/L[Final]
2L Guba-Straub (GS)7.0 KCl 2.5M 120 ml300mM
K2HPO4 1.0M 50 ml 50mM
KH2PO4 1.0M 100 ml100mM
4L 0.4% NaHCO3 (w/v)8.5 NaHCO3 solid 42 g
6L Cold dH2O
16L Cold Acetone (spectral grade)
Method:
1.)Pick-up the rabbit from animal resources (117 Jackson) and carry it up to the wetlab in the rabbit box. Place the box in a large green plastic bag. Insert the CO2 and run a gentle flow of CO2 into the bag for 5, which should be enough time to asphyxiate the rabbit. Hold the dead rabbit over the sink and slit the throat with a sharp scalpel. Squeeze as much blood as possible out of the rabbit over the sink. Lay the rabbit down on the covered benchtop and completely remove the skin from the torso and legs. Make an incision through the stomach muscles and completely gut the rabbit, including trimming out any fat. Rinse the carcass out with cold H2O over the sink and then put it in a tray of ice. Cover the carcass with more ice and let it chill for 20 in the coldbox. This should all be done as quickly as possible. The pelt, all unwanted tissue, and the carcass should all be discarded in a large green plastic bag. Once done with the carcass, tie the bag closed and dispose of it in the animal disposal room (61 Lyon). All subsequent steps should be carried out in the coldbox (4C) or on ice unless otherwise specified.
2.)Trim the external fascia off of the back and leg muscles of the rabbit, then dissect out the white muscle from the back and legs. Keep the muscle on ice. Trim off the fat, fascia, and any red muscle from the white muscle and cut it into small pieces.
3.)Grind the muscle into rabbit burger using the meat grinder and weigh it in a large plastic weigh boat.
Total Weight:
4.)Transfer the rabbit burger into a 4L beaker. Add 1L of GS and extract the homogenate 10 to remove the myosin, stirring occasionally. Dilute the homogenate with an equal volume (1-2L) of cold dH2O and allow it to sit in the coldbox for 5 so the residue can settle to the beaker bottom and won’t clog the cheesecloth. Squeeze the homogenate through 2 layers of cheesecloth (into a funnel over a large beaker). Discard the liquid (contains myosin).
5.)Return the residue to the large beaker, scraping off the cheesecloth if necessary. Add 2L of cold dH2O and stir for 5. Let the residue settle for 5 and then squeeze it through 2 layers of cheesecloth again. Discard the liquid.
6.)Wash the residue twice with 2L 0.4% NaHCO3 as above. The first wash is for 10 with stirring and the second wash is for 5 with stirring. For both washes let the residue settle for 2 before squeezing it through the cheesecloth. Discard the liquid from both washes.
7.)Wash the residue twice as quickly as possible (stir for a few seconds and then squeeze through the cheesecloth) with 1L of cold dH2O (or the actin is extracted). Discard the liquid.
8.)Wash the residue with 1-2L of cold acetone for 10 with occasional stirring. Squeeze through 2 layers of cheesecloth and discard the used acetone. Repeat the acetone wash 5-8 times until the discarded acetone remains clear upon the addition of al equal volume of H2O.
9.)Allow the resulting acetone powder to dry overnight at room temperature under a hood between 2 layers of cheesecloth (with weights on the sides).
10.)Weight the dried acetone powder into a tarred plastic bottles. Store in the 0C walk-in freezer labeled with name, date and weight. Determine the final weight and yield (g acetone powder/g muscle) of the prep.
Final Weight:Yield:
Comments:
Fajerlab under EDITMethods.doc10/20/1999 4:55 PM10/20/1999 4:55 PM