First of all it would be useful to have the precise protocol you used. Tubulin purification is a rather streamlined approach and easy to follow, although it requires a lot of preparations before prep-day. Important things for tubulin purification from mammalian brain sources are: use freshly collected brains. From slaughter site to lab brains must be washed with and be stored under ice-cold PBS. Frozen-stored brains dont work. Before arriving at the lab centrifuges, rotors, buffers and columns must be already at hand from the day before. In the cold room you must remove meninges and blood clots as thoroughly and as fast as possible and then process according to standard protocols (i.e., http://mitchison.med.harvard.edu/protocols.html). The provided web page gives a straight forward and fail proof procedure. Read it carefully, follow instructions and good luck

Hi George,

I have already purified tubulin from goat brains (purified by double cycling step). During extraction they seemed to be active and were immediately snapped into liq. nitrogen (and stored at -80). The problem arises when I am trying to polymerize them in smaller aliquots (~13ul) with DMSO, MgCl2, BRB80 and GTP. They just dont polymerize..!!!

I have reprepared every reagent and have varied the concentrations and time of polymerization but nothing seems to work...

What concentrations of tubulin, DMSO, MgCl2, BRB80, and GTP are you using? What temperature are you doing the polymerization at?

tubulin - 65uM

MgCl2 - 4mM

GTP - 1mM

DMSO - 5%

BRB80 - 0.9ul

these are final concentrations in the polymerization mixture. The total reaction volume is 12.5 ul.

I polymerize them at 37 degree celsius.

You might find this protocol useful. When I've done microtubule polymerization in the past, I never added extra MgCl2. Your tubulin and GTP concentrations are in the right range. Is your tubulin stock stored in BRB80? You may want to try using 10% DMSO.

http://mitchison.med.harvard.edu/protocols/microtubules/Tubulin%20Polymerization%20with%20GTP.pdf

My tubulin is stored in BRB80 AT -80 degree celsius.

I did go through this protocol earlier but couldn't get anything. I will try with 10% DMSO though. I also doubt if my tubulin is in the correct concentration perhaps I will need to recalibrate the spectrophotometer.

What length of microtubules did you get after polymerization, with respect to time?

Hi Sankalp

If your tubulin (but I think it is microtubule since you talked about 2 cycles and didn't mention any purification step) is preserved properly in active state, you need only 10-15 uM tubulin. You can use 1mM MgSO4 (no need of 4 mM) and 1mM EGTA in addition to the BRB80 buffer. 8% DMSO (very good quality) should work. Be careful to minimize the in between times when the protein is not frozen. Keep everything precooled in the cold steps. I think this should work. Good luck.

Hi Suparna,

Thanks for the suggestion but the issue got resolved long back.

I am polymerizing it at 4ug/ml and use glycerol instead.

Thanks again.

hi Sankalp

Would you mind sharing the detailed condition you use for the final successful polymerization? And what improvements make your system work? That would be a great favor, I come across the same problem recently:when polymerizing at 37℃ using the turibidity method, the tubulin I purified didn't seem to be active, and the change in OD340 was just 0.1（%>_

Hi Nan,

What is the final concentration of tubulin that you use for polymerization?

From what I could guess, you are diluting 4ug/ml stock to a lower concentration in 100ul BRB80.

Try increasing the concentration of tubulin and keep it above 1mg/ml. Also, you can polymerize tubulin for 20 min (instead of 1 hour) and spin them down at around 120000 xg to see if you get a microtubule pellet (free tubulin wont pellet).

If your GTP gets exhausted, your microtubule would depolymerize back unless stabilized by paclitaxel or any other stabilizing molecule.

Please let me know if it works.

hi Sankalp,

Thanks for answing me.

Sorry I didn't make myself clear, the final concentration of tubulin is 4ug/ul(or 4mg/ml, about 40uM) in my 100ul system, I did the experiment in half-area 96well plate according to Cytoskeletion manul,except that I use homemade tubulin instead of purchased protein.(http://www.cytoskeleton.com/bk004p)So, i think the concentration may not be the failure factor,I just couldn't get it work.

Your suggestion that check the polymerizaiton condition after 20min is quite useful,I can use that to check my reagents using small scale of reaction,thank you.

Add, if you don't mind,I really want to know how it turn to work in your experiment?what adjustment did you do? I doubt that the inappropriate reaction buf would make the experiment fail...

http://www.cytoskeleton.com/bk004p

Hi Nan,

Try finding out the presence of microtubules using any other characterization technique (you can try centrifuging it to see the presence of any pellet and then run SDS PAGE of that pellet).

In my case, I was using TRITC labeled tubulin mix with unlabeled tubulin and was trying to see them using a fluorescence microscope. My labeled tubulin got denatured so I couldn't see them under the microscope. Though the unlabeled tubulin was polymerizing (I confirmed it by centrifuging the polymerized solution).

Also, do check if your GTP is working and increase the concentration of tubulin just to account for tubulin denatured during purification. You can even use paclitaxel to stabilize your microtubules.

In fact there can be a lot of factors, like presence of Calcium etc, which are taken care of by BRB80. 

hi Sankalp.

Thank you for sharing,I will try again,taking your advices into consideration.

I will let you know if it finally works out.

Lot thanks!!