重组蛋白纯化说明书解析.docx

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