蛋白质定量分析自聚合沉降速率法.pdfVIP

QuantitativeAnalysisofProtein

Self-AssociationbySedimentation

Velocity

HuayingZhao,1WenqiLi,2WendanChu,2MaryBollard,1ReginaAdão,1

andPeterSchuck1,3

1DynamicsofMacromolecularAssemblySection,LaboratoryofCellularImagingand

MacromolecularBiophysics,NationalInstituteofBiomedicalImagingand

Bioengineering,NationalInstitutesofHealth,Bethesda,Maryland

2NationalProteinScienceFacility,SchoolofLifeScience,TsinghuaUniversity,Beijing,

China

3Correspondingauthor:schuckp@

Sedimentationvelocityanalyticalultracentrifugationisapowerfulclassical

methodtostudyproteinself-associationprocessesinsolutionbasedonthesize-

dependentmacromolecularmigrationinthecentrifugalield.Thistechnique

canelucidatetheassemblyscheme,measureafinitiesrangingfrompicomolar

tomillimolarKd,andinfavorablecasesprovideinformationonoligomerlife-

timesandhydrodynamicshape.Thepresentstep-by-stepprotocolsdetailthe

essentialstepsofinstrumentcalibration,experimentalsetup,anddataanalysis.

Usingawidelyavailablecommercialproteinasamodelsystem,theprotocols

invitereplicationandcomparisonwithourresults.Acommentarydiscusses

principlesformodiicationsintheprotocolsthatmaybenecessarytooptimize

applicationofsedimentationvelocityanalysistootherself-associatingproteins.

©2020WileyPeriodicalsLLC.

BasicProtocol1:Measurementofexternalcalibrationfactors

BasicProtocol2:Sedimentationvelocityexperimentforproteinself-

association

BasicProtocol3:SedimentationcoeficientdistributionanalysisinSEDFIT

andisothermanalysisinSEDPHAT

Keywords:bindingisotherminstrumentcalibrationproteinself-association

sed