TheTdp1AssayKitcontainsallreagentsforroutinedetectionofTdp1activityusingthetyrosinereleaseassayfromthesubstrateOligo-Tyr(humantdp1issoldseparately). TheassayishighlyspecificforTdp1.
Summary:
ThekitcontainsreagentsandprotocolsforassayingTyrosylDNAPhosphodiesterase(Tdp1). Aprocedureformakingsuitablecellextractsisdescribedattheendofthismanual.
KitContents:
Volumesaregivenforthe100assaykitsize. Forthe250assaykitsize,multiplyvolumesby2.5fold. NotethatthiskitdoesnotincludeTyrosylDNAPhosphodiesterase,whichissoldseparately(seelinkabove). WerecommendreconstructingtheassayusingthepurifiedTdp1toensurethattheassayisoptimizedandworkingwell.
- Oligo-Tyrsubstrate (10ulat0.8ug/ulinTEbuffer[10mMTris,pH7.5,1mMEDTA]). ThissubstrateshouldbeendlabeledwithT4polynucleotidekinaseandg-[32P]-ATPasdescribedinthekitprotocol
- 5xTdp1assaybuffer(300ul): 5xbuffercontains100mMTris-HCl(pH7.5),500mMKCl,50mM EDTA,5mM dithiothreitol(DTT).
- 2xStopBuffer(gelloADIngbuffer)(200ul):96%formamide,20mMEDTA,0.03%xylenecyanol,0.03%bromophenolblue.
AssayProtocolOverview:
Reactionvolumesshouldbe10-20ulfinalvolume(limitedbyvolumethatcanbeloadedontothegel). Reactionsareassembledinmicrofugetubeswithwater,bufferandOligo-Tyrosinesubstrate. Thetestfractionsshouldbeaddedlastandthereactionsincubatedat37Cforatleast15min.thenterminatedwith10ulofthestopbuffer. AnalyzeonadenaturingpolyacrylamidegelorbyDNAsequencinggelelectrophoresis.
ActualTdp1assayisshownbelow. Inthisanalysis,5’endlabeledOligo,linkedtoatyrosinemoietyontheopposite(3’)end,wasincubatedwithpurifiedhumanTdp1fromTopoGEN. ThereleaseoftyrosinewasmeasuredonaDNAsequencinggelthatmonitorsachangeinmolecularweight.
