MIME-Version: 1.0 Content-Location: file:///C:/65743233/Removalofmicrotubuleassociatedproteins.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Removal of microtubule associated proteins

Removal of microtubule associated proteins

There are a number of methods for removing MAP= s from MTP to produce purified tubulin: DEAE-Sephadex or phosphocellulose chromatography, or cycling in the presence of high concentrations of NaGlutamate or NaPIPES. <= span style=3D'mso-spacerun:yes'> High salt concentrations remove MAPs from MTs.  We find high = NaPIPES cycling is rapid, simple and provides both high yields and activity. <= /o:p>

 

1.&n= bsp;     Thaw a pellet of twice-cycled MTP and using a cutoff large tip remove 0.5 ml (the remainder can be refrozen in liquid nitrogen a= nd stored at –80o) and transfer to a 1.5 ml Eppendorf tube.  Adjust the MTP to O.5M = PIPES, 1mMMgCl2, 1mM GTP, 10% dimethyl sulfoxide (DMSO) by addition of 0.55 ml 1M PIPES, pH = 6.9, 12 microliters 100mM MgCl2, 12 microliters 100mM NaGTP a= nd 0.12 ml dimethyl sulfoxide (DMSO).  Vortex gently, but thoroughly.

2.&n= bsp;     Incubate at 37o for 10 minuts in a water bath. The solution should become very turbid and viscous.

3.&n= bsp;     Pellet MTs at 18,000 rp= m in a Sorvall SS-34 rotor for 20 minutes or in a Beck= man TLA 100.3 at 50,000 rpm (k=3D47) for 5 minutes at 37o.  Rotors should be pre-warmed to 37<= sup>o.

4.&n= bsp;     Remove the supernatant that contains the MAPs. The pellet is MAP-depleted tubulin.