b'Figures 58 show example data from experiments using various isolation strategies.MS Bead MS BeadLoad elution boil Load elution boilDynabeads Protein G Cytiva Protein G Bio-Rad QiagenDynabeads Protein G Cytiva Protein GMilliporeSigmaPP2A (72%) -catenin (140%)60 min 10 min 60 min 10 min PTEN (90%) HDAC1 (140%)P13K (247%)EGFR (80%)Stat1 (88%) pan Akt (97%)25 kDaFigure 5. Protein yields determined by western blot analysis.Figure 7. The Thermo Scientific Pierce MS-Compatible Magnetic Invitrogen Dynabeads Protein G magnetic beads have the best overallIP Kit (streptavidin) allows for effective target capture and elution. performance for yield, capacity, and nonspecific binding. Percentages indicate elution efficiency compared to bead boil. The eluates were analyzed by western blot. Antibodies were labeled with the Thermo Scientific Pierce Antibody Biotinylation Kit for IP and used with the Pierce MS-Compatible Magnetic IP Kit to immunoprecipitate target proteins from cell lysates.ThermoScienticU2OS Pierce Cytiva MilliporeSigmacelllysate A/G G A G A47.7 31.5 Cdk1 (~34 kDa)L FT E BB FT E BB FT E BB24.4Thermo Scientic MilliporeSigmaFigure 6. Higher IP yield with Protein A/G beads. U2OS (humanFigure 8. Comparison of protein purification using osteosarcoma) cells were lysed in IP Lysis/Wash Buffer, and incubatedThermo Scientific Pierce Anti-DYKDDDDK Affinity Resin and with and without anti-Cdk1 antibody overnight at 4C. Thermo Scientificanother suppliers product. C-terminal DYKDDDDK-tagged GFP Pierce Protein A/G Magnetic Beads were compared to Mag Sepharoseprotein was expressed using the Thermo Scientific 1-Step Human Beads (Cytiva) and PureProteome Protein A and Protein G productsHigh-Yield Maxi In Vitro Translation (IVT) Kit and immunoprecipitated (MilliporeSigma). The beads were washed multiple times using ausing Pierce Anti-DYKDDDDK Affinity Resin or Anti-FLAG M2 Affinity KingFisher Flex instrument and then eluted with SDS-PAGE reducingGel (MilliporeSigma). Tagged proteins were competitively eluted sample buffer for 10 minutes at room temperature. The eluates werewith Pierce 3x DYKDDDDK Peptide and analyzed by western blot. resolved by SDS-PAGE and analyzed by western blot for Cdk1. Comparison of the starting lysate (L), flow-through (FT), eluate (E), and bead-boiled sample (BB) shows effective capture and elution of DYKDDDDK-tagged proteins.To learn more about other magnetic bead products to help isolate everything from cells to exosomes, download the Dynabeads magnetic beads brochure at thermofisher.com/dynabeadsProtein purification thermofisher.com/proteinbiology 37'