Viscosity Polymeriztion of Microtubules 实验方法 丁 ...
来自 : 蚂蚁淘
LEVELII
Materials
- Tubulin(BrainextractfromExercise9.4)
- GTP
- ATP
- Viscometer
- Waterbathorincubatorat37°C
Procedure
- ComputetheamountofGTP(M.W.523)neededtomakeafinalconcentration0.5mMGTPwhenaddedto40ml.ofbrainextractinmicrotubulebuffer.Likewise,computetheamountofdryATP(M.W507)neededtomakeafinalconcentrationof0.5mMATP.
- Withthebrainextractinanicebath,addboththeGTPandtheATP(swirltodissolve)andimmediatelytransferthemixturetoawaterbathat37°C.
- Useaviscometertomeasuretheviscosityat3minuteintervalsforaperiodof30minutes,oruntilchangeisnolongerobserved.
Aviscometerisadevicewhichmakesuseofcapillaryflow.Insertaglassviscometerintoawaterbathattheappropriatetemperature(37°Cforpolymerization)andadda3.0mlsampletotheviscometer.Applysuctionorpressureasappropriatetotheviscometertopullthesampleintotheexpandedsamplechamber.Releasethesuction(orpressure)andmeasurethetimeittakesforthesampletomovefromtheuppermarkontheviscometertothelowermark.
Usesucrosesolutionsofknownviscositytocalibratetheviscometer.
- Plotthechangeinviscositywithtimeofincubation.
Notes
TubulinandMAP"swillpolymerizeinthepresenceofMg
andGTP.Thetubulinextractisplacedinabufferthatsuppliesallofthenecessaryingredients,exceptGTP.UpontheadditionofGTP,thetubulinwillbegintopolymerizeandcanbemonitored.
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