重组蛋白纯化说明书解析.docx
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重组蛋白纯化说明书解析.docx
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重组蛋白纯化说明书解析
InstructionManual
ProBondTMPurificationSystem
Forpurificationofpolyhistidine-containingrecombinantproteins
Catalognos.K850-01,K851-01,K852-01,K853-01,K854-01,R801-01,R801-15
VersionK
2September2004
25-0006
ii
TableofContents
KitContentsandStorage..................................................................................................................................ivAccessoryProducts...........................................................................................................................................viIntroduction.................................................................................................................................1Overview..............................................................................................................................................................1Methods.......................................................................................................................................2PreparingCellLysates........................................................................................................................................2PurificationProcedure—NativeConditions....................................................................................................7PurificationProcedure—DenaturingConditions.........................................................................................11PurificationProcedure—HybridConditions.................................................................................................13Troubleshooting.................................................................................................................................................15Appendix....................................................................................................................................17AdditionalProtocols.........................................................................................................................................17Recipes................................................................................................................................................................18FrequentlyAskedQuestions............................................................................................................................21References...........................................................................................................................................................22TechnicalService...............................................................................................................................................23
iii
KitContentsandStorage
TypesofProductsThismanualissuppliedwiththefollowingproducts:
ProductCatalogNo.ProBond™PurificationSystemK850-01
ProBond™PurificationSystemwithAntibody
withAnti-Xpress™AntibodyK851-01
withAnti-myc-HRPAntibodyK852-01
withAnti-His(C-term-HRPAntibodyK853-01
withAnti-V5-HRPAntibodyK854-01
ProBond™Nickel-ChelatingResin(50mlR801-01
ProBond™NickelChelatingResin(150mlR801-15
ProBond™PurificationSystemComponentsTheProBond™PurificationSystemincludesenoughresin,reagents,andcolumnsforsixpurifications.Thecomponentsarelistedbelow.Seenextpageforresinspecifications.
ProBond™Resin50%slurryin20%ethanol12ml5XNative
PurificationBuffer
250mMNaH2PO4,pH8.0
2.5MNaCl
1×125mlbottle
GuanidiniumLysis
Buffer
6MGuanidineHCl
20mMsodiumphosphate,pH7.8
500mMNaCl
1×60mlbottle
Denaturing
BindingBuffer
8MUrea
20mMsodiumphosphate,pH7.8
500mMNaCl
2×125mlbottles
DenaturingWash
Buffer
8MUrea
20mMsodiumphosphate,pH6.0
500mMNaCl
2×125mlbottles
DenaturingElution
Buffer
8MUrea
20mMNaH2PO4,pH4.0
500mMNaCl
1×60mlbottle
Imidazole3MImidazole,
20mMsodiumphosphate,pH6.0
500mMNaCl
1×8mlbottle
Purification
Columns
10mlcolumns6
Continuedonnextpage
iv
KitContentsandStorage,Continued
ProBond™PurificationSystemwithAntibodyTheProBond™PurificationSystemwithAntibodyincludesresin,reagents,andcolumnsasdescribedfortheProBond™PurificationSystem(previouspageand50µloftheappropriatepurifiedmousemonoclonalantibody.Sufficientreagentsareincludedtoperformsixpurificationsand25Westernblotswiththeantibody.
Formoredetailsontheantibodyspecificity,subclass,andprotocolsforusingtheantibody,refertotheantibodymanualsuppliedwiththesystem.
StorageStoreProBond™resinat+4°C.Storebufferandcolumnsatroomtemperature.Storetheantibodyat4°C.Avoidrepeatedfreezingandthawingofthe
antibodyasitmayresultinlossofactivity.
Theproductisguaranteedfor6monthswhenstoredproperly.
Allnativepurificationbuffersarepreparedfromthe5XNativePurificationBufferandthe3MImidazole,asdescribedonpage7.
TheDenaturingWashBufferpH5.3ispreparedfromtheDenaturingWashBuffer(pH6.0,asdescribedonpage11.
ResinandColumn
Specifications
ProBond™resinisprechargedwithNi2+ionsandappearsblueincolor.Itisprovidedasa50%slurryin20%ethanol.
ProBond™resinandpurificationcolumnshavethefollowingspecifications:
•BindingcapacityofProBond™resin:
1–5mgofproteinpermlofresin•Averagebeadsize:
45–165microns
•Poresizeofpurificationcolumns:
30–35microns
•Recommendedflowrate:
0.5ml/min
•Maximumflowrate:
2ml/min
•Maximumlinearflowrate:
700cm/h
•Columnmaterial:
Polypropylene
•pHstability(longterm:
pH3–13
•pHstability(shortterm:
pH2–14
Product
Qualification
TheProBond™PurificationSystemisqualifiedbypurifying2mgofmyoglobinproteinonacolumnandperformingaBradfordassay.Proteinrecoverymustbe75%orhigher.
v
AccessoryProducts
Additional
Products
ThefollowingproductsarealsoavailablefororderfromInvitrogen:
ProductQuantity
CatalogNo.
ProBond™Nickel-ChelatingResin50ml
150ml
R801-01R801-15
Polypropylenecolumns
(empty
50R640-50
Ni-NTAAgarose10ml25mlR901-01R901-15
Ni-NTAPurificationSystem6purificationsK950-01Ni-NTAPurificationSystem
withAntibody
withAnti-Xpress™AntibodywithAnti-myc-HRPAntibodywithAnti-His(C-term-HRPAntibody
withAnti-V5-HRPAntibody1kit
1kit
1kit
1kit
K951-01K952-01K953-01K954-01
Anti-mycAntibody50µlR950-25Anti-V5Antibody50µlR960-25Anti-Xpress™Antibody50µlR910-25Anti-His(C-termAntibody50µlR930-25InVision™His-tagIn-gelStain500mlLC6030InVision™His-tagIn-gel
StainingKit
1kitLC6033
Pre-CastGelsandPre-madeBuffersAlargevarietyofpre-castgelsforSDS-PAGEandpre-madebuffersforyourconvenienceareavailablefromInvitrogen.Fordetails,visitourwebsiteatorcontactTechnicalService(page23.
vi
Introduction
Overview
IntroductionTheProBond™PurificationSystemisdesignedforpurificationof6xHis-taggedrecombinantproteinsexpressedinbacteria,insect,andmammaliancells.ThesystemisdesignedaroundthehighaffinityandselectivityofProBond™Nickel-ChelatingResinforrecombinantfusionproteinscontainingsixtandemhistidineresidues.
TheProBond™PurificationSystemisacompletesystemthatincludes
purificationbuffersandresinforpurifyingproteinsundernative,denaturing,orhybridconditions.Theresultingproteinsarereadyforuseinmanytargetapplications.
Thismanualisdesignedtoprovidegenericprotocolsthatcanbeadaptedforyourparticularproteins.Theoptimalpurificationparameterswillvarywitheachproteinbeingpurified.
ProBond™Nickel-ChelatingResinProBond™Nickel-ChelatingResinisusedforpurificationofrecombinantproteinsexpressedinbacteria,insect,andmammaliancellsfromany6xHis-taggedvector.ProBond™Nickel-ChelatingResinexhibitshighaffinityandselectivityfor6xHis-taggedrecombinantfusionproteins.
Proteinscanbepurifiedundernative,denaturing,orhybridconditionsusingtheProBond™Nickel-ChelatingResin.ProteinsboundtotheresinareelutedwithlowpHbufferorbycompetitionwithimidazoleorhistidine.Theresultingproteinsarereadyforuseintargetapplications.
BindingCharacteristicsProBond™Nickel-ChelatingResinusesthechelatingligandiminodiaceticacid(IDAinahighlycross-linkedagarosematrix.IDAbindsNi2+ionsbythreecoordinationsites.
Theprotocolsprovidedinthismanualaregeneric,andmaynotresultin100%pureprotein.Theseprotocolsshouldbeoptimizedbasedonthebindingcharacteristicsofyourparticularproteins.
NativeVersus
Denaturing
Conditions
Thedecisiontopurifyyour6xHis-taggedfusionproteinsundernativeordenaturingconditionsdependsonthesolubilityoftheproteinandtheneedtoretainbiologicalactivityfordownstreamapplications.
•Usenativeconditionsifyourproteinissoluble(inthesupernatantafterlysisandyouwanttopreserveproteinactivity.
•Usedenaturingconditionsiftheproteinisinsoluble(inthepelletafterlysisorifyourdownstreamapplicationdoesnotdependonproteinactivity.
•Usehybridprotocolifyourproteinisinsolublebutyouwanttopreserveproteinactivity.Usingthisprotocol,youpreparethelysateandcolumnsunderdenaturingconditionsandthenusenativebuffersduringthewashandelutionstepstorefoldtheprotein.Notethatthisprotocolmaynotrestoreactivityforallproteins.Seepage14.
1
Methods
PreparingCellLysates
IntroductionInstructionsforpreparinglysatesfrombacteria,insect,andmammaliancellsusingnativeordenaturingconditionsaredescribedbelow.
MaterialsNeededYouwillneedthefollowingitems:
•NativeBindingBuffer(recipeisonpage8forpreparinglysat
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