Agrisera/HSP70/HSC70 | Heat shock protein 70/AS09 592/


productinformation
Background		Heat-shockprotein70(Hsp70)isthemajorstress-inducibleproteininvertebratesandhighlyconservedthroughoutevolution.Itplaysaroleasamolecularchaperoneandisimportantforallowingcellstocopewithacutestressorinsult,especiallythoseaffectingtheproteinmachinery.Akillifishisalargefamilyof variousoviparous(egg-laying)cyprinodontiformfishincluding1270differentspecies.
Immunogen		KLH-conjugatedC-terminalsyntheticpeptideconservedinhsc/hsp70sequencesfromawiderangeofanimalspecies
Host		Rabbit
Clonality		Polyclonal
Clone
Purity		Serum
Format		Lyophilized
Quantity		200µl
Reconstitution		Forreconstitutionadd200µlofsterilewater.
Storage		storelyophilized/reconstitutedat-20°C;oncereconstitutedmakealiquotstoavoidrepeatedfreeze-thawcycles.Please,remembertospintubesbrieflypriortoopeningthemtoavoidanylossesthatmightoccurfromlyophilizedmaterialadheringtothecaporsidesofthetubes.
Testedapplications		westernblot(WB)
Relatedproducts		collectionofantibodiestofishrelatedtargets Secondaryantibodies
Additionalinformation		Chosenpeptidesequenceisalsoconservedinseveralfishspeciesincluding:DQ202278.1HSC70Fundulus,DQ202279.1HSP70-1Fundulus,DQ202280.1HSP70-2Fundulus,BT059361.1HSC70Salmosalar(atlanticsalmon),AB092839.2HSP70Carassiusauratus(goldfish),BC056709.1HSP70Daniorerio(zebrafish)andotheranimalHSP70proteins

applicationinformation
Recommendeddilution		1:10000withECLAdvance(WB)
Expected\|apparentMW		70kDa
Confirmedreactivity		Arabidopsisthaliana,Acanthamoebacastellanii(amoeba),Dictyosteliumdiscoideum,frog-heart,Frog-skeletalmuscle,Frog-liver,Caenorhabditiselegans,salmon(Salmosalar),rainbowtrout(Oncorhynchusmykiss),cow,chicken,pig,rat,seal,mummichog
Predictedreactivity		atlanticsalmon(Salmosalar),brooktrout(Salvelinusfontinalis)
Notreactivein		noconfirmedexceptionsfrompredictedreactivityknowninthemoment
Additionalinformation		notavailableatthemoment
Selectedreferences		Chandraetal.(2012).Sustainedhightemperatureincreasesthevitellogeninresponseto17alpha-ethynylestradiolinmummichog(Fundulusheteroclitus).Aquatictoxicology.

applicationexample

Dictyosteliumdiscoideum(1),Acanthamoebacastellanii(2),Frog-heart(3),Frog-skeletalmuscle(4),Frog-liver(5),Caenorhabditiselegans(6),Arabidopsisthaliana(7),tissueswerehomogenizedinglasshomogenizerinPBSbufferandcentrifugedat500xgfor5min.Supernatantwascollectedand50µgofproteinforeachgellanewasdenaturedwithLaemmlibufferat950Cfor5min.Sampleswereseparatedon14%SDS-PAGEandblotted1htonitrocelluloseusingsemi-drytransfer.Blotwasblockedovernightin5%milkinTBSbufferandnextincubatedintheprimaryantibodyatadilutionof1:2000for1hatRTwithagitation.Theantibodysolutionwasdecantedandtheblotwaswashedoncefor15minand4timesfor5mininTBS-TatRTwithagitation.Blotwasincubatedinsecondaryantibody(anti-rabbitIgGhorseradishperoxidaseconjugated,from)dilutedto1:25000for1hatRTwithagitation.Theblotwaswashedasaboveanddevelopedfor5minwithRocheBMChemiluminescenceWesternBlottingKit.Exposuretimewas300seconds.

CourtesyDr.MałgorzataSłocińska,UAM,Poland

western blot detection of HSP70 invarious animaltissues

5µgoftotalproteinfrom(1)cowmuscle,(2)chickenmuscle,(3)pigmuscle,(4)ratliver,(5)salmonmuscle,(6)sealmuscle,(7)mummichogheatshockcontrol,(8)mummichogheatshockpost-24hours,extractedwithProteinExtrationBuffer,PEB(AS08300),wereseparatedon4-12%NuPage(Invitrogen)LDS-PAGEandblotted1htoPVDF.Blotswereblockedimmediatelyfollowingtransferin2%ECLAdvanceblockingreagent(GEHealthcare)in20mMTris,137mMsodiumchloridepH7.6with0.1%(v/v)Tween-20(TBS-T)for1hatroomtemperaturewithagitation.Blotswereincubatedintheprimaryantibodyatadilutionof1:10000for1hatroomtemperaturewithagitation.Theantibodysolutionwasdecantedandtheblotwasrinsedbrieflytwice,thenwashedoncefor15minand3timesfor5mininTBS-Tatroomtemperaturewithagitation.Blotswereincubatedinsecondaryantibody(Agriseraanti-rabbitIgGhorseradishperoxidaseconjugated,AS09602)dilutedto1:50000in2%ECLAdvanceblockingsolutionfor1hatroomtemperaturewithagitation.Theblotswerewashedasaboveanddevelopedfor5minwithECLAdvancedetectionreagentaccordingtothemanufacturersinstructions.ImagesoftheblotswereobtainedusingaCCDimager(FluorSMax,Bio-Rad)andQuantityOnesoftware(Bio-Rad).Exposuretimewas30seconds.

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Agrisera/HSP70/HSC70 | Heat shock protein 70/AS09 592/