Protein characterization by static light scattering.pdf

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1、中国试剂网2.4.21ProteincharacterizationbystaticlightscatteringIntroductiontostaticlightscatteringStaticlightscattering(SLS)isanon-invasivetechniquewherebyanabsolutemolecularmassofaproteinsampleinsolutionmaybeexperimentallydeterminedtoanaccuracyofbetterthan5%throughexposuretolowintensitylaserlight(6

2、90nm).Theintensityofthescatteredlightismeasuredasafunctionofangleandmaybeanalyzedtoyieldthemolarmass,rootmeansquareradius,andsecondvirialcoefficient(A).TheresultsofanSLS2experimentscanbeusedasaqualitycontrolinproteinpreparation(e.g.forstructuralstudies)inadditiontothedeterminationofsolutionoli

3、gomericstate(monomer/dimeretc.).SLSexperimentsmaybeperformedineitherbatchorchromatographymodes.However,asthemeasurementyieldsthevolume-averagedmolecularweightofthesamplewithinthelaserbeamitismorepowerfultoutilizethetechniqueincombinationwithproteinpurification.Asthemeasurementsareperformedinaf

4、lowcellthereisnolossofsampleandtheSLSdetectorcaneasilybeintegratedintostandardproteinpurificationequipment.Duetothenecessityofobtaininggoodbaselinesinboth280nmabsorptionmeasurements(UV)andlightscattering(LS)measurements,SECrepresentsagoodchoiceofseparationmedia,duetotheuseofonlyasinglebuffersy

5、stemfortheentirepurification.Sincethelightscatteringandconcentrationaremeasuredforeachelutingfraction,themassandsizecanbedeterminedindependentlyoftheelutionposition.Thisisparticularlyimportantforproteinspecieswithnon-globularshapes,whichmayeluteatpositionsdistantfromthatpredictedbythecalibrati

6、oncurveforthecolumn.Materials1.Proteinsolution(s)tobeanalyzed(seeNotes1and2).2.ÄktaPurifier(GEHeathcare)appliedwithasizeexclusionchromatographycolumn(seeNotes3and4).3.Staticlightscatteringdetector(WyattTechnologyminiDAWNTristar)(seeNote5).4.BSAsolution(10mg/mlinwater)Experimentalset-upandcalib

7、ration1.ForSLSmeasurementsinthechromatographymodetheminiDAWNTristar(SLSdetector)shouldbeplacedintheflowpathoftheÄktaPurifier.TheinstrumentistypicallyequippedwithaSuperdex20010/300GLSECcolumn.TheSLSdetectorusestheUVsignalfromtheÄktaPurif