ubpbio/Rapid 26S Proteasome Purification KitL/J4320/1 Kit

TheRapid26SProteasomePurificationKitisdesignedtorapidlypurifytheendogenous26Sproteasomefromcellortissueextractswithin8hoursaccordingtoapublishedmethod(reference1)withmodifications.ThisapproachusestheN-terminalubiquitin-like(Ubl)domainofhumanRAD23Basanaffinitybait,whichallowstherapidandgentleisolationofendogenous26Sproteasomes.Thebound26SproteasomeissubsequentlyelutedusingtheC-terminaltwoubiquitin-interactingmotifs(UIM)ofhumanS5a.

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Thiskitissufficientforpurificationofthe26Sproteasomefrom300mg,6X50mgor30X10mgcellortissueextractsataconcentrationof5mg/ml.Usually,1-2%oftotalcellularproteinsarethe26Sproteasome.
Description
TheRapid26SProteasomePurificationKitisdesignedtorapidlypurifytheendogenous26Sproteasomefromcellortissueextractswithin8hoursaccordingtoapublishedmethod(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)
j4310image

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)

ProductName:

Rapid26SProteasomePurificationKit-L

AlsoKnownAs:

N/A

CatalogNo.:

J4320

Size:

1Kit

MolecularWeight:

N/A

Species:

N/A

Source:

N/A

Stock:

Enzymestockedin20mMTris,150mMNaCl,2mMβME,10%Glycerol;SubstratestockedinDMSO

Concentration:

Seetubelabel

QualityAssurance:

Enzyme>95%bySDS-PAGE;Substrate>98%byHPLCandNMR

Storage:

Storeat-80°C;avoidmultiplefreeze-thawcycles

PDFDataSheet:

PDFdatasheet,MSDS

NCBIRefSeq:

N/A

Image(s):

(Clickimagetoenlarge)

Schematicof26Sproteasomeenrichment

ShippingMethod:

Dryiceshipping

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.

Schematicof26Sproteasomeenrichment

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ubpbio/Rapid 26S Proteasome Purification KitL/J4320/1 Kit

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