ProductName: | Rapid26SProteasomePurificationKit-L |
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AlsoKnownAs: | N/A |
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CatalogNo.: | J4320 |
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Size: | 1Kit |
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MolecularWeight: | N/A |
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Species: | N/A |
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Source: | N/A |
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Stock: | Enzymestockedin20mMTris,150mMNaCl,2mMβME,10%Glycerol;SubstratestockedinDMSO |
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Concentration: | Seetubelabel |
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QualityAssurance: | Enzyme>95%bySDS-PAGE;Substrate>98%byHPLCandNMR |
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Storage: | Storeat-80°C;avoidmultiplefreeze-thawcycles |
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PDFDataSheet: | PDFdatasheet,MSDS |
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NCBIRefSeq: | N/A |
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Image(s): | (Clickimagetoenlarge)
Schematicof26Sproteasomeenrichment | |
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ShippingMethod: | Dryiceshipping |
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References: | 1.BescheHC,HaasW,GygiSP,GoldbergAL.Biochemistry.2009;48:2538-49. 2.ElsasserS,SchmidtM,FinleyD.MethodsEnzymol.2005;398:353-63. 3.KisselevAF,GoldbergAL.MethodsEnzymol.2005;398:364-78.
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Thiskitissufficientforpurificationofthe26Sproteasomefrom300mg,6X50mgor30X10mgcellortissueextractsataconcentrationof5mg/ml.Usually,1-2%oftotalcellularproteinsarethe26Sproteasome.
DescriptionTheRapid26SProteasomePurificationKitisdesignedtorapidlypurifytheendogenous26Sproteasomefromcellortissueextractswithin8hoursaccordingtoapublishedmethod(reference1)withmodifications.ThisapproachusestheN-terminalubiquitin-like(Ubl)domainofhumanRAD23Basanaffinitybait,whichallowstherapidandgentleisolationofendogenous26Sproteasomes.Thebound26SproteasomeissubsequentlyelutedusingtheC-terminaltwoubiquitin-interactingmotifs(UIM)ofhumanS5a.
Components•GST-Ubl(4mg/ml)...................................................2X1.2ml•GST(4mg/ml).........................................................2X1.2ml
•6xHis-UIM(4mg/ml)....................................................1X1ml•20XLysisBuffer........................................................3X1.2ml
•NaCl(1M)...............................................................2X1.25ml•ATP(500mM).................................................................0.5ml
•SUC-LLVY-AMC(50mM)..................................................30µl•GlutathioneAgaroseResin(75%slurry)...........2X1.0ml
•NickelXPureAgaroseResin(50%slurry)..........2X500µl
Images(Clickimagetoenlarge)
A.CoomassiestainedSDS-PAGEof5μgpurifiedGST-Ubl.B.CoomassiestainedSDS-PAGEof5μgpurified6xHis-UIM.C.CoomassiestainedSDS-PAGEof3μgpurifiedhuman26Sproteasomes.
D.Coomassiestainednative-PAGEof3μgpurifiedhuman26Sproteasomes.E.Solutionpeptidaseassayofthepurifiedhuman26Sproteasome.150μlreactionmixturescontained10nMpurifiedhuman26Sproteasomein50μMSUC-LLVY-AMCin20mMTris,pH7.1at37°C,2mMμME.ThereleasedAMCfluorescencewasmeasuredusingaplatereaderwithexcitationandemissionfiltersat360/40and460/30nm,respectively.Thisassaywasusedtomonitorthechymotrypsin-likeactivityofthe26Sproteasome.(Clickimagetoenlarge)
Schematicof26Sproteasomeenrichment |
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