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Agrisera/RbcL II | Rubisco form II positive control/quantitation standard/AS15 2955S/
productinformation
Background

Rubisco(Ribulose-1,5-bisphosphatecarboxylase/oxygenase)catalyzestherate-limitingstepofCO2fixationinphotosyntheticorganisms.FormIIRubiscoispresentinmanyphotosyntheticbacteriaandarchaeaandinsomephotosyntheticdinoflagellates.

SourceofRubiscostandard:OverexpressedRbcLformIIprotein

Host
Clonality
Clone
Purity
Format
Quantity
Reconstitution
Storage

storelyophilized/reconstitutedat-20°C;oncereconstitutedmakealiquotstoavoidrepeatedfreeze-thawcycles.Please,remembertospintubesbrieflypriortoopeningthemtoavoidanylossesthatmightoccurfromlyophilizedmaterialadheringtothecaporsidesofthetubes.

Testedapplicationswesternblot(WB)
Relatedproducts

PrimaryantibodywhichismatchingRbcLformIIstandard:

AS152955 |anti-RbcL TypeII,rabbitglobalantibody

collectionofotherproteinstandards

collectionofglobalantibodies

Plantandalgalproteinextractionbuffer

Additionalinformation

TheRbcLproteinstandardcanbeusedinacombinationwithAgriseraglobalantibiodies(AS152955fromrabbit)toquantitateRbcLfromawiderangeofspecies.Globalantibodiesareraisedagainsthighlyconservedaminoacidsequence.Thisstandardisalsoincludedinfollowingkits:Educationalantibodykit-photosynthesis, PhotosynthesisToolKit-quantitation,Rubicoquantitationkit,

Quantitativewesternblot: detailedmethoddescription,videotutorial

applicationinformation
Recommendeddilution

Standardcurve:3loadsarerecommended(0.5,2and4μl).
Formostapplicationsasampleloadof0.2μgofchlorophyll/wellwillgiveaRbcLsignalinthisrange.

Positivecontrol:a2μlloadperwellisoptimalformostchemiluminescentdetectionsystems.HigherstandardconcentrationneedstobeusedtoallowdetectionbyCoomasiestains.Suchgelswithhigherstandardconcentrationcannotbeusedforquantitationusingchemiluminescence.

ThisstandardisstABIlizedandreadyanddoesnotrequireheatingbeforeloADIngonthegel.

Pleasenotethatthisproductcontains10%glycerolandmightappearasliquidbutisprovidedlyophilized.Allowtheproductseveralminutestosolubilizeafteraddingwater.MixthoroughlybutgentlyTakeextracaretomixthoroughlybeforeeachuse,astheproteinstendtosettlewiththemoredenselayerafterfreezing.

Expected|apparentMW

52.7kDa

Confirmedreactivity
Predictedreactivity
Notreactivein
Additionalinformation

Concentration:afterre-constitutionwithsterilemilliQwaterfinalconcentrationofthestandardis0.15pmoles/µl

Proteinstandardbuffercomposition:Glycerol10%,TrisBase141mM,TrisHCl106mM,LDS2%,EDTA0.51mM,SERVA®BlueG2500.22mM,PhenolRed0.175mM,pH8.5,0.1mg/mlPefaBlocproteaseinhibitor(Roche),50mMDTT.

Thisstandardisready-to-loadanddoesnotrequireanyadditionsorheating.Itneedstobefullythawedandthoroughlymixedpriortousing.Avoidvigorousvortexing,asbufferscontaindetergent.Followingmixing,brieflypulseinamicrocentrifugetocollectmaterialfromcap.
Thisstandardisstabilizedandreadyanddoesnotrequireheatingbeforeloadingonthegel.
Pleasenotethatthisproductcontains10%glycerolandmightappearasliquidbutisprovidedlyophilized.Allowtheproductseveralminutestosolubilizeafteraddingwater.MixthoroughlybutgentlyTakeextracaretomixthoroughlybeforeeachuse,astheproteinstendtosettlewiththemoredenselayerafterfreezing.

Please,usethe55kDasizeofRbcLforcalculations.ThepmolesinthestandardrefertopmolesofrbcLmonomers.

Selectedreferencestobeaddedwhenavailable,productreleasedinSeptember2015.

applicationexample


western blot using anti-RbcL form II antibodies

1.5µgoftotalproteinextractfromRhodobactercapsulatus(1);extractedwithProteinExtractionBufferPEB(AS08300);0.5pmolofrecombinantRbcLI(2),0.5pmolofrecombinantRbcLII,product AS152955S(3)Samplesweredilutedwith1Xsamplebuffer(NuPAGELDSsamplebuffer(Invitrogen)supplementedwith50mMDTTandheatat70°Cfor5minandkeeptonicebeforeloading.Proteinsampleswereseparatedon4-12% BoltPlusgels,  LDS-PAGEandblottedfor 70minutes toPVDFusingtanktransfer.Blotswereblockedimmediatelyfollowingtransferin2%blockingreagent(GERPN2125;Healthcare)or5%non-fatmilkdissolvedin20mMTris,137mMsodiumchloridepH7.6with0.1%(v/v)Tween-20(TBS-T)for1hatroomtemperaturewithagitation.Blotswereincubatedintheprimaryantibodyatadilutionof1:10000(inblockingreagent)for1hatroomtemperaturewithagitation.Theantibodysolutionwasdecantedandtheblotwasrinsedbrieflytwice,andthenwashed1x15minand3x5minwithTBS-Tatroomtemperaturewithagitation.Blotswereincubatedinsecondaryantibody(anti-rabbitIgGhorseradishperoxidaseconjugated,recommendedsecondaryantibody AS101489,Agrisera)dilutedto1:25000inblockingreagentfor1hatroomtemperaturewithagitation.Theblotswerewashedasabove.Theblotwasdevelopedfor5minwithTMA-6(lumigen)detectionreagentaccordingthemanufacturersinstructions.ImagesoftheblotswereobtainedusingaCCDimager(VersaDocMP4000) andQuantityOnesoftware(Bio-Rad).Exposuretimewas30seconds.


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