Viscosity&PolymeriztionofMicrotubules

LEVELII

Materials

• Tubulin(BrainextractfromExercise9.4)
• GTP
• ATP
• Viscometer
• Waterbathorincubatorat37°C

Procedure

1. ComputetheamountofGTP(M.W.523)neededtomakeafinalconcentration0.5mMGTPwhenaddedto40ml.ofbrainextractinmicrotubulebuffer.Likewise,computetheamountofdryATP(M.W507)neededtomakeafinalconcentrationof0.5mMATP.

2. Withthebrainextractinanicebath,addboththeGTPandtheATP(swirltodissolve)andimmediatelytransferthemixturetoawaterbathat37°C.

3. Useaviscometertomeasuretheviscosityat3minuteintervalsforaperiodof30minutes,oruntilchangeisnolongerobserved.

   Aviscometerisadevicewhichmakesuseofcapillaryflow.Insertaglassviscometerintoawaterbathattheappropriatetemperature(37°Cforpolymerization)andadda3.0mlsampletotheviscometer.Applysuctionorpressureasappropriatetotheviscometertopullthesampleintotheexpandedsamplechamber.Releasethesuction(orpressure)andmeasurethetimeittakesforthesampletomovefromtheuppermarkontheviscometertothelowermark.

   Usesucrosesolutionsofknownviscositytocalibratetheviscometer.

4. Plotthechangeinviscositywithtimeofincubation.

Notes

TubulinandMAP"swillpolymerizeinthepresenceofMgandGTP.Thetubulinextractisplacedinabufferthatsuppliesallofthenecessaryingredients,exceptGTP.UpontheadditionofGTP,thetubulinwillbegintopolymerizeandcanbemonitored.

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