Microtubule Binding Assays相关交流-蚂蚁淘在线

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=V_maxandm2=K_d

Notes

1.	Motorbindingtomicrotubulesissensitivetoionicstrength,whichshouldbeoptimizedtobehighenoughtopreventmotoraggregationwithoutpreventingbindingtomicrotubules.Fortyto50mMNaCl+buffergivesgoodresultsforseveralmotorproteins,includingNcdandKar3.

2.	Performassaysonmutantandwild-typemotorsonthesamedayusingthesamesolutionsandmicrotublesformoreaccuratecomparisons.

================ 蚂蚁淘在线 ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容