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Microtubule sedimentation assay - (Feb/25/2009 )

Hi,

I'm trying to do a microtubule sedimentation assay, but all the protocols I have get indicate different concentrations of polymerized microtubules. Has anyone done this assay before? Thanks!!

-Jess24-

Hi jess, can you email me some of your protocols? I want see if we are doing the same thing.
shawnixes@hotmail.com

thanks

Jess24 on Feb 25 2009, 01:31 PM said:

Hi,

I'm trying to do a microtubule sedimentation assay, but all the protocols I have get indicate different concentrations of polymerized microtubules. Has anyone done this assay before? Thanks!!

-shawnee-

Hello guys,

If its not confidential, could you please share it here. Let others learn as well. I am curious too what you are doing. (Frankly, heard this thing for the first time and I am working with microtubules as well)

-noelmathur-

noelmathur on Feb 26 2009, 08:17 AM said:

Hello guys,

If its not confidential, could you please share it here. Let others learn as well. I am curious too what you are doing. (Frankly, heard this thing for the first time and I am working with microtubules as well)


The microtubule sedimentation assay (aka microtubule binding assay):

I'm using commercial tubulin (1mg/mL). I'm polymerizing the tubulin adding taxol stepwise. Using the following protocol:

Tubulin Polymerization:

1. On ice, mix tubulin in 1X BRB80 with 1 mM DTT and 1 mM GTP. Incubate at 0C for 5min.
2. Clarify the mix in TLA100 rotor at 90K rpm for 5min at 2C. Incubate the supernatant at 37C for 1-2min.
3.Add taxol stepwise (equimolar ratios below) as follows (for 1 mg/ml tubulin):
Add 1/10 vol 1 μM taxol; Incubate at 37C for 5-10min
Add 1/10 vol 10 μM taxol; Incubate at 37C for 5-10min
Add 1/10 vol 100 μM taxol; Incubate at 37C for 15min
3. Pellet microtubules over a warm 40% glycerol in BRB80 cushion in a TLA100, 100.2 or 100.3 rotor, 12min at cca. 200,000xg (cca 70krpm for any of them).
4. Discard the supernatant.
5. Rinse the pellet with warm (37C) water (preferably with 0.5% Triton X 100).
6. Resuspend in warm BRB80 + 1 mM DTT + 10-20 μM taxol (taxol should be at least equimolar and preferably in excess to the tubulin)

For the actual sedimentation assay:

I'm using different concentrations of tubulin (0-5uM) and 200ng GST fusion proteins in a 40 uL reaction. The reactions are incubated for 30 min at 37C to allow binding to occur, and then centrifuged as above to pellet the microtubules. Pellets and supernatants are fractionated on SDS-PAGE gels and probed by immunoblotting with anti-GST antibodies.

-Jess24-