Protein characterization by static light scattering

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1、ProteincharacterizationbystaticlightscatteringIntroductiontostaticlightscatteringStaticlightscattering(SLS)isanon-invasivetechniquewherebyanabsolutemolecularmassofaproteinsampleinsolutionmaybeexperimentallydeterminedtoanaccuracyofbetterthan5%throughexposuretolowintensitylaserlight(690n

2、m).Theintensityofthescatteredlightismeasuredasafunctionofangleandmaybeanalyzedtoyieldthemolarmass,rootmeansquareradius,andsecondvirialcoefficient(A).TheresultsofanSLS2experimentscanbeusedasaqualitycontrolinproteinpreparation(e.g.forstructuralstudies)inadditiontothedeterminationofsoluti

3、onoligomericstate(monomer/dimeretc.).SLSexperimentsmaybeperformedineitherbatchorchromatographymodes.However,asthemeasurementyieldsthevolume-averagedmolecularweightofthesamplewithinthelaserbeamitismorepowerfultoutilizethetechniqueincombinationwithproteinpurification.Asthemeasurementsare

4、performedinaflowcellthereisnolossofsampleandtheSLSdetectorcaneasilybeintegratedintostandardproteinpurificationequipment.Duetothenecessityofobtaininggoodbaselinesinboth280nmabsorptionmeasurements(UV)andlightscattering(LS)measurements,SECrepresentsagoodchoiceofseparationmedia,duetotheuse

5、ofonlyasinglebuffersystemfortheentirepurification.Sincethelightscatteringandconcentrationaremeasuredforeachelutingfraction,themassandsizecanbedeterminedindependentlyoftheelutionposition.Thisisparticularlyimportantforproteinspecieswithnon-globularshapes,whichmayeluteatpositionsdistantfr

6、omthatpredictedbythecalibrationcurveforthecolumn.Materials1.Proteinsolution(s)tobeanalyzed(seeNotes1and2).2.ÄktaPurifier(GEHeathcare)appliedwithasizeexclusionchromatographycolumn(seeNotes3and4).3.Staticlightscatteringdetector(WyattTechnologyminiDAWNTristar)(seeNote5).4.BSAsolution(10mg

7、/mlinwater)Experimentalset-upandcalibration1.ForSLSmeasurementsinthechromatographymodetheminiDAWNTristar(SLSdetector)shouldbeplacedintheflowpathoftheÄktaPurifier.TheinstrumentistypicallyequippedwithaSuperdex20010/300GLSECcolumn.TheSLSdetectorusestheUVsignalfromtheÄktaPurifiertomeasur