Numerouscellularfunctionsoccurinspatiallyandtemporallyconfinedregions.Recentstudieshaveshownthatmembrane-lessorganellesandcompartmentsinthecellareassembledvialiquid-liquidphaseseparation(LLPS).InvitroLLPSassaysusingrecombinantexpressedandpurifiedproteinsarenecessaryforustofurtherunderstandhowtheassemblyofphase-separatedcompartmentsisregulatedincells.However;uniformstandardsandprotocolsarelackingfortheseinvitrostudies.Here,wedescribeastep-by-stepprotocolcommonlyusedtoinvestigateinvitroLLPSusingpurifiedproteins.Thisprotocolincludesexpressionandpurificationofthestudiedproteins,inductionofLLPSofthepurifiedproteins,andstudiesofthebiophysicalpropertiesoftheliquiddropletsformedbyLLPS.TheseprotocolscanbeeasilyfollowedbyresearcherstoinvestigatetheLLPSbehaviorsofproteinsofinterest.
