Materials

Siliconizedultracentrifugemicrofugetubes

GTP-depletedmicrotubules

6XSDSloADIngdye

1XSDSloadingdye

CoomassieBrilliantBlueR250(0.8%in50%methanol+10%aceticacid)

Destainingsolution(13%methanol+3%aceticacid)

Procedure

1.	Mixbufferandtaxolinasiliconizedultracentrifugemicrofugetube,thenaddmotorandGTP-depletedmicrotubulesusingasetconcentrationofmicrotubules(e.g.,2µM)andarangeofmotorconcentrations(e.g.,0.5-10µM).Clickhereforanexampleofasetofassays.

2.	Incubate5minatroomtemperature.

3.	Centrifugeat50,000rpmfor20minat22°C.Duringcentrifugation,prepare1tubeforeachassayandadd5µL6XSDSloadingdyetothetube.

4.	Carefullyremovetheupper20µLofeachsupernatantandplaceintotubecontaining6XSDSloadingdye.

5.	Removeremainingsupernatantanddiscard.

6.	Add30µL1XSDSloadingdyetopelletandresUSPendbyvortexingvigorously.

7.	Boilsamples2-3minandloadequalamounts(e.g.,5µL)ontoanSDS-PAgel.Supernatantsfromassayscontaininggreaterthan2µMmotorcanbeloadedathalfofthevolumeinsteadofthefullvolumeforgreateraccuracyduringquantitation.

8.	StaingelwithCoomassieBluefor1houranddestainfor2-3hours.

9.	Scangelsintodigitalimagesusingaconventionalscanner(e.g.,AgfaArcusIIscanner)oradedicatedgelscanner.Thegelcanbeplacedonatransparentyellowsheetforscanningtoreducecontrast.

10.	Quantitatethebound(pellet)andunbound(supernatant)motorforeachassayusingNIHImage(clickheretoobtainacopy).

11.

AnalyzethedatausingaprogramsuchasKaleidaGraph(SynergySoftware),correctingtheboundmotorandtotalconcentrationofmotorforthemotorprecipitatedwithoutaddedmicrotubules.Plottheconcentration(µM)ofunboundversusboundmotor,andfitthedatapointswiththeMichaelis-Mentonequationwherey=boundmotorandx=unboundmotorineachassay: y=m1*x/(m2+x) m1=Vmaxandm2=Kd Notes 1. Motorbindingtomicrotubulesissensitivetoionicstrength,whichshouldbeoptimizedtobehighenoughtopreventmotoraggregationwithoutpreventingbindingtomicrotubules.Fortyto50mMNaCl+buffergivesgoodresultsforseveralmotorproteins,includingNcdandKar3. 2. Performassaysonmutantandwild-typemotorsonthesamedayusingthesamesolutionsandmicrotublesformoreaccuratecomparisons.