Cy3 labeling of tubulin
Tubulin is labeled in polymeric form to protect residues that are important
for assembly. Tubulin can be successfully conju=
gated
with fluorescent derivatives of amine-reactive succini=
midyl
esters. Functional tubulin is selected by cycle=
s of
polymerization/ depolymerization. By modifying =
the
ratio of reactants, the Cy3 labeling protocol has also been used to derivatize tubulin with C=
y3.5,
Cy5, tetramethylrhodamine, X-rhodamine,
Oregon Green and Alexa 488 and Alexa
568 succinimidyl esters.
1.&n= bsp; Estimate the tubulin pe= llet volume from Step 4 above. The pellet volume will be much less than the orig= inal MTP pellet volume as removal of MAPs that proje= ct from the walls of MTs allows closer packing. The protein concentration of MAP-depleted MT pellets is about 100 mg ml-1<= /sup>.
= 1. The polymer formed in 0.5M PIPES with 10% DMSO is in the form of tubulin sheets rather than closed tubules. Therefore polymer is depolymerized and reassembled in normal MT polymerization buffer to prevent possible modification of portions of the molecule internal to the MT.
2.&n= bsp; Resuspend the pellet in= 2 volumes of ice cold PM buffer (0.1 M PIPES, pH 6.9, 1 = mM MgCl2) by pipeting up and= down with a cutoff pipette tip. It is important to keep the tubulin concentration high to push the subsequent reaction.
3.&n= bsp; Incubate on ice for 10 minutes.
4.&n= bsp; Adjust the solution to 1 mM GTP and 10% DMSO. Tubulin lacking MAPs assembles poorly, but addition of DMSO greatly enhances assembly.
5.&n= bsp; Incubate at 37o for 10 minutes to polyme= rize MTs.
6.&n=
bsp;
Dissolve 2 vials of Cy3 reactive dye (Amersham Pharmacia Biotech,
7.&n= bsp; Incubate for 40 minutes at 37o.
8.&n=
bsp;
Pellet the labeled MTs =
at
18,000 rpm in a Sorvall SS-34 rotor for 20 minu=
tes or
in a Beckman TLA100.3 at 50,000 rpm for 5 minutes at 37o. Rotor should be pre-warmed to 37
9.&n= bsp; Carefully remove the supernatant. Estimate the volu= me of the pellet and resuspend in 5 volumes of ice= cold PM as in Step 2.
10.&= nbsp; Incubate on ice for 10 minutes
11.&= nbsp; Centrifuge at 18,000 rpm for 20 minutes at 4o<= /sup> in a Sorvall SS-34 rotor or a Beckman TLA-100.3= rotor at 50,000 rpm for 5 minutes at 4o. Rotor should be pre-cooled to= 4o.
12.&=
nbsp;
Carefully remove the supernatant and adjust to 1
13.&= nbsp; Incubate 10 minutes at 37o.
14.&=
nbsp;
Pellet the labeled MTs =
at
18,000 rpm in a Sorvall SS-34 rotor for 20 minu=
tes or
in a Beckman TLA100.3 at 50,000 rpm for 5 minutes at 37o. Rotor should be pre-warmed to 37
= 15. Carefully remove the supernatant. Estimate the volume of the pellet and resuspend in 5 volumes of ice cold PM buffer as in St= ep 6.
16.&= nbsp; Incubate on ice for 10 minutes.
17.&= nbsp; Centrifuge at 18,000 rpm for 20 minutes at 4o<= /sup> in a Sorvall SS-34 rotor or a Beckman TLA-100.3= rotor at 50,000 rpm for 5 minutes at o. Rotor should be pre-cooled to = 4o.
18.&= nbsp; Carefully remove the supernatant and determine its = tubulin concentration and labeling stoichiometry by measuring the absorbance of a 1/100 dilution in PM buffer at 280 nm and = at 556 nm (Cy3 peak). The extinction coefficient of tubul= in at 280 nm is 80,000 M-1cm-1, for Cy3 at 556 nm is 150= ,000 M-1cm-1 and the contribution of Cy3 to absorbance at = 280 nm is 0.08 times its absorbance at 556 nm. Labeling st= oichiometry is typically 0.3-0.6 Cy3/tubulin.
= 19. Adjust the concentration of Cy3-tubulin to 10 mg ml-1 (100 micromolar) with PM buffer= .
= 20. Freeze ten microliter aliquots in liquid nitrogen. The preparation yields 50-80 aliqu= ots.
Solutions:
PEM: 1 l =3D NaPIPES, 33.7 g, EGTA 0.38 g, MgCl2 10 ml of 100 mM<= /span> stock; titrate to pH 6.9.
PM: 100 ml= =3D NaPIPES 3.37 g, MgCl2 1.0 ml of 100 mM stock; titrate to pH 6.9.
100 mM= Na 3 GTP: 1 ml =3D 0.060 g
100 mM= MgCl2 hexahydrate: 100 ml =3D 2.03 g=