MT Assembly

Microtubule Assembly



    Materials


      PC – Purifed tubulin (alpha/beta tubulin MW = 100,000)


      PM = 100 mM
      2 mM
      1 mM
      		 PIPES, pH 6.9
      EGTA
      Mg2SO4


      4 mM Taxol (MW = 853.9) in DMSO


      50 mM Mg-GTP =

      		 50 µl 100 mM GTP, pH ~7
      50 µl 100 mM Mg2SO4


    Procedure







      1.
      		 Thaw frozen tubulin by swirling in 37°C waterbath.

      Adjust concentration to 40 µM with PM.

      Add 50 mM Mg-GTP to 1 mM.

      Mix gently.

      Incubate 30 minutes at 37°C.
      2.
      		 Warm equal volume of 40 µM taxol diluted in PM to 37°C.

      Add in 2-3 aliquots to tubulin + GTP, mix gently.

      Incubate 5 minutes at 37°C.
      3.
      		 Aliquot MTs and flash freeze in liquid nitrogen.

      Final = 2 mg/ml (20 µM) tubulin/MTs

      0.5 mM Mg-GTP

      20 µM taxol

      1 X PM buffer



    Modified from T. Salmon Laboratory



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Created 2 March 1999 21:00 GMT

Modified 27 March 2002 18:55 GMT