浙江|安徽|四川|云南化工产品供应商
来自 : 蚂蚁淘
byChang-DukJun,03/14/2000
Purpose
Materials
- Antibody7E3,2Lsupgrowninflasks,frozenandthawedovernight.
- BioRadAffi-GelProteinAMAPSIIBufferscat.#1530-6160($161.00)
- 50mMTrispH7.8
- 40gammoniumsulfateforevery100mlSup.
- 4L100mMTris/pH7.8orBindingBuffer
- BDtubing14-170-12F(Fisher)
- PolyethylenetubingClayAdanes.
- 18Gaugeneedles.
- BindingBuffer(BioRad):make1L.314g/LddH2Oandfilterthrough0.22um,filter.pH=9.0
- Elutionbuffer(BioRad):make500ml.11g/500mlddH2Oandfilterasbefore.pH=3.0
- Regenerationbuffer(BioRad):BioRadAffi-gelregenerationbuffer.
Procedure
ForAmmoniumsulfatecut:- FilterSup.in1LCostarFilterusingpre-filter.
- Add50mMTrispH7.8(50mlof1Mstock/Liter).100ml
- Add40gammoniumsulfateforevery100mlSup.(slowly).800g
- StirO/Nincoldroom.
- Pourintoplasticbottles.Spinat4oC,7,500rpmfor20mininJA-10rotor.
- Prepare4L100mMTris/pH7.8(400mlof1Mstock/4L)orBindingbuffer.
- DiscardSup.ResUSPendpelletsin10mlof100mMTrisbufferorBindingbuffer.Andpoolinto50mlFalcontubes(trynottomakebubbles).
- Use3.2ml/cm12-14,000MWdialysistubing.
- Heattubingin500mlH2Oinmicrowave.Notboiling.
- RinsetubinginfreshH2Oseveraltimes.
- Testeachtubew/H2Oanddiscard.
- Addproteinmixturetodialysistube.Stirslowlyin100mMTrisbufferorBindingbufferuntilpinkcolorisout.
- Transferproteinmixture(Ab)to50mlFalcontubestodeterminevolume.
- Dilutethemixture1:1withBindingbuffer.
- Filterthrough0.45umfilters(useprefilters).
ForPurificationofAbsbyProteinAcolumn:- PrepareProteinAcolumn.
- Runbindingbuffer(pH9.0)~200ml.
- Filterproteinmixture
- AddproteinmixtureorculturesupernatantcontainingAb(adjustpHto7.8withBindingbuffer;redcolor)totheProteinAcolumn.
MouseantibodiesoftheIgG1subclassdonothaveahighaffinityforproteinA.PurificationonproteinAbeadsusingstandardconditionswillyieldapproximately1/10theamountofantibodycomparedwithothersubclasses.IncaseofIgG1subclass,add3.3MNaCl(192.85g/L)tocrudeantibodypreparation(serum,tissueculturesupernatant,orascites).
- ApplyBindingbufferagain~200ml.
- ApplyElutionbuffer~100ml.
- Collect3mlfractionsin5mltubeswith700ul1MTrispH9.0alreadyinbottomoftubetoneutralize(collectat~5min/fraction).
- 25tubesaresufficientforcollection.Ingeneral,youcanseehighAbconcentrationsin7-8tubes.
- Test1ulonpHpaper.
- ReadODtoknowAbconcentrations.
- OD(Absorbanceat280nm)/1.35=Xmg/ml.
- StoreAbsat-20oCorfurtherconcentratebyusingCentriprep.Andstoreat-20oC.
- RegenerateAbcolumnwithRegeneratebuffer(~200ml).
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