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        Making Microtubule Seeds

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        相关专题
         
        DMSO
          Prechill glycerol, MgCl2, and GTP on ice. Rapidly thaw frozen tubulin, then immediately place on ice. Unpolymerized tubulin is labile!
          200 µL aliquot of tubulin (cycled)--in BRB80, stored in -70℃
          100 µL glycerol
          10 µL 0.1 M MgCl2
          3 µL 0.1 M GTP (pH adjusted to neutral)
          or
          50 µL aliquot of tubulin (cycled)--in BRB80, stored in -70℃
          25 µL glycerol
          2.5 µL 0.1 M MgCl2
          0.75 µL 0.1 M GTP (pH adjusted to neutral) Add other ingredients. Mix gently, but thoroughly. Spin tube briefly. Aliquot tubulin to small (500 µL) tubes that have been prechilled on ice. A standard titration of binding activity requires ~15 µLs. Flash freeze aliquots in liquid nitrogen and store at -70℃. To make polymerized MTs: Place "seeds" at 37℃ for 30 min. Add taxol to 10 µM. I add the required amount of taxol in 1 µL (i.e. for a 12 µL aliquot of MT seeds, add 1 µL of 0.13 mM taxol in ). Mix in gently. Incubate an additional 15 min. at 37℃. Taxol-stabilized MTs are stable at room temp. for several days.
          NOTE: To dilute MTs, make a cocktail similar to that which the seeds are in:
          200 µL BRB80
          100 µL glycerol
          10 µL 0.1 M MgCl2
          Adjust small aliquots to 10 µM taxol prior to use. Making Long Microtubules
          100 µls Tubulin (3-5 mg/ml)
          3.3 µls 100 mM MgCl2
          1.3 µls 100 mM GTP
          2 µls seeds 45 minutes at 37℃ Add Taxol ~7µls 15 minutes at 37℃


          8/25/95
          NEW Protocol for Assembling Microtubules
          •To a tube containing 10µls of 10mg/ml tubulin add: 10µls G-PEM with 30% Glycerol; 0.75 µl of rhodamine labelled tubulin
          •Place at 35℃ for 10"
          •remove to room temp.
          •Immediately add paclitaxel (taxol) to 10µM final (2µls of a 0.1 mM stock taxol into 20µls)
          •the microtubules formed by this method have a mean length of ~2µm and can be stable for upto several weeks at room temp.

          G-PEM buffer :
          80mM Pipes
          1mM MgCl2
          1mM EGTA
          1mM GTP pH 6.8 - 7.0
          10% Glycerol
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