MIME-Version: 1.0 Content-Location: file:///C:/2A4BB24E/Microtubuleproteinpreparation.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Microtubule protein preparation

Microtubule protein prepa= ration

Purification of microtub= ule protein (MTP) is based on the temperature dependence of tubulin polymerization.  MTP is prepar= ed from brain tissue by two cycles of temperature–dependent polymerizati= on-depolymerization. Each cycle selects for assembly com= petent tubulin. Besides tubulin, twice-cycled MTP contains a large fraction of co-purifying proteins designa= ted microtubule associated proteins (MAPs) that are removed prior to fluorescent labeling.

        &= nbsp;   Our lab's tubulin prep is a vintage one as the orig= inal reference is Borisy&nb= sp; et al.,  1975. M= inor modifications have been made over the past three decades. We use a specially constructed flow-through teflon-stainless steel homogenizer, but a Waring = blendor and/or motor driven Potter-Elvehjem tissue grin= der can substitute.  It is necessa= ry to have access to two Sorvall RC-5B centrifuges (or equivalent) set to 4o and 37o for temperature cycling= . If two sets of Sorvall GSA or SS-34 rotors are not available, rotors can be rapidly brought to the proper temperature by incubating in ice or water baths.

 

Note: To allow substitution for ultracentrifuge rotors at equivalent sedimentation conditions, the k factor= is provided for the designated rotor.

 

= 1.      Obtain 24 hog brains from a slaughterhouse. It is essential that the brains are fr= esh (best if obtained within 20 minutes of slaughter- they should still feel wa= rm). The brains should be immediately immersed in ice for transport to the lab.<= /p>

= 2.      Remove superficial blood clots and vessels from brains.  This is simply accomplished by blo= tting with KimWipes.

= 3.      Dissect the cortex away from underlying white matter. Each brain yields about 30 g = of cortex. Weigh cortices. Our usual preparation uses 600 g of cortex.        =            

4.&n= bsp;     Homogenize 5-10 seconds in a p= rechilled Waring blendor in a= cold room with 1.33 volumes  (800 m= l) ice cold PEM (0.1M piperazine-N,N-bis(2-ethanesulfo= nic acid) (PIPES), 1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N,N,-= tetraacetic acid (EGTA), 1 mM MgCl2, 0.1 mM guanosine 5' -triphosphate (GTP), pH 6.9) plus 0.1 mM GTP.

5.&n= bsp;     Transfer in batches to a cold 200-250 ml Potter-Elvehjem tissue grinder with a motor driven pestle and homogenize with 2-3 passes at 2000 rpm in a cold room.

1.&n= bsp;     Note: = Good yields can also be obtained by omitting Step 4 and homogenizing as in Step 2 except for 3 x 15 seconds.

6.&n= bsp;     Transfer the homogenate to 6 250 ml polycarbonate <= st1:City w:st=3D"on">Oak Ridge style b= ottles and centrifuge in a Sorvall GSA rotor (cooled t= o 4o) at 12,500 rpm for 90 minutes at 4o.

= 2.      Alternatively, centrifugation for 15 minutes at 12,500 rpm in a Sorva= ll GSA rotor, followed by centrifugation for 60 minutes in a Beckman Type 21 r= otor at 20,000 rpm (k=3D440) can be used.

= 7.      Decant the supernatant into a graduate cylinder and measure the volume.  Transfer to a flask, add solid GTP= to 1 mM (0.06 g/100 ml) and swirl to dissolve.

8.&n= bsp;     Dispense the supernatant into 250ml bottles and incubate at 37o for 45 minutes in a water bath with shaker.  An increase in turbidity and visco= sity as evidenced by air bubble trapping will occur as MTs<= /span> assemble.

9.&n= bsp;     Centrifuge in a Sorvall= GSA rotor at 12,500 rpm for 45 minutes at 37o (rotor pre-warmed to 3= 7o).

= 10.   Resuspend the gelatinous pellets in 1/8 volume of the original supernatant (from Step 6) with ice cold PEM + 1 mM GTP using a large-bore pipette. Homogenize with 2-3 passes with a motor dri= ven Potter-Elvehjem homogenizer at 2000 rpm.

11.&= nbsp;  Incubate on ice for 30 minutes to depolymerize MTs.

12.&= nbsp;  Centrifuge in a Sorvall= SS-34 rotor at 18,000 rpm for 30 minutes at 4o.

3.&n= bsp;     This constitutes one cycle of polymerization-depolymerization.

13.&= nbsp;  Remove the supernatant, transfer to 12ml polycarbon= ate tubes and incubate at 37o for 30 minutes in a water bath with shaker. The solution will become very turbid and viscous.

14.&= nbsp;  Centrifuge in a Sorvall= SS-34 rotor (pre-warmed to 37o) at 18,000 rpm for 30 minutes at 37o.

15.&= nbsp;  Carefully aspirate the supernatant. MT pellets can = be quite loose.  The yield can be estimated given that MTP pellets contain about 25 mg ml-1 of protein. The yield of MTP is typically (0.6-1 mg of MTP per 1 g of cortex).=

16.&= nbsp;  Freeze the pellets in liquid nitrogen and store at –80o.