MIME-Version: 1.0 Content-Location: file:///C:/086BB1F1/Isolationofactin1.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Isolation of actin1

Isola= tion of actin1

 

1.&n= bsp;     Add 20 ml of 0o buffer A (2mM Tris-Cl, 0.2 mM adenosine 5'-triphosphate (ATP), 0.5 mM dithiothreitol (DTT), 0.2 mM CaCl2, 1 mM Na N= 3) per gram acetone powder (typically 10 g) and extract with stirring on ice f= or 30 minutes.

2.    Centrifuge the mixture at 16,000 rpm in a Sorv= all SS-34 rotor for 30 minutes at 4o. Filter the supernatant through 2 lay= ers of cheesecloth to remove any floating material.

Optiona= l: re-extract the pellet with the original volume buffer and repeat Step 2.

3.    Measure the volume of the (combined if re-extracted) supernatant and add KCl to 50 mM (from 3M stock) and MgCl2 to 2 mM (from 100 mM stock) while stirring.

4.    Leave 30 minutes at room temperature. As the a= ctin polymerizes, the viscosity should increase as indicated by trapping of air bubbles.

5.    Cool on ice.

6.   Slowly add solid KCl to 0.8 = M (5.6 gm/100 ml) with stirring at 4o. This step dissociates tropomyosin from F-actin.

7.    After 30 minutes, centrifuge for 2 hours in an ultracentrifuge rotor at a speed such that the k factor is less than 250, e= .g. Beckman Type 45Ti at 35,000 rpm, k=3D220. Under centrifugation conditions w= ith lower k factor the time of centrifugation can be reduced proportionately.

8.    Discard the supernatant leaving a clear/translucent gelatinous pellet. Gently rinse the pellet with 2 ml of Buffer A.

9.    Remove the pellet from the tube with a spatula= and by scraping the remainder into a small volume of Buffer A.

10.  Re-suspend the pellet in 3 ml of Bu= ffer A per original gram of acetone powder using a Potter-Elvehjem homogenizer. The suspension should be very viscous.

11.  Transfer to thin dialysis tubing (1= 0-20 mm diameter) and dialyze against 20 volumes of Buffer A) for 2 days with 3 changes of buffer. This will depolymerize greater than 90% of actin filamen= ts.

12.=   Centrifuge as for Step 7.

13.  The supernatant is purified G-actin= . The expected yield is 10-30 milligrams of actin per gram of acetone powder. Act= in concentration can be determined by measuring absorbance at 290 nm and using= an extinction coefficient of 0.63 ml/milligram-cm.

14.  G-actin solutions are stable for on= ly 2-3 days at 4o.  G-acti= n in 5 mM Tris (pH=3D8.0), 1 mM ATP, 1 mM DTT can be frozen in liquid nitrogen and stored at –80o where it remains active for more than a yea= r.

 

Solutions:

Buffer A:

        =             &nb= sp;            =          Stock        =             &nb= sp;            =             &nb= sp;            =          1 liter

2mM Tris-Cl        =             &nb= sp; 1.0 M=3D 12.1 gm Tris-base/ 100 ml (pH=3D8.0)      2 ml

0.2 mM Na2ATP        =         0.1 M= =3D 6.05 gm Na2ATP- 3 H2O/ 10 ml        =     2 ml

0.5 mM DTT        =             &nb= sp; dry        =             &nb= sp;            =             &nb= sp;            =              77 mg

0.2 mM CaCl2        =             1.0 M =3D 14.7 gm CaCl2 - 2 H2O/ 100 ml        =     0.2 ml

1 mM NaN3        =             &nb= sp;   1.0 M=3D 6.3 gm/ 100 ml        =             &nb= sp;            =       1 ml

titrate to pH=3D8.0<= o:p>

 

3 M KCl=3D 22.4 gm/100 ml

100 mM MgCl2 hexahydrate =3D 2.03 gm/100 ml

 

Note: Actin is very susceptible to proteolysis resulting from bacterial contamination. All stock solutions should be stored at 4o with 0.1% sodium azide.

 

1 This protoco= l is modified from J.A. Spudich and S. Watt, J. Biol. Chem. 246: 4866 (1971).