For immunoprecipitation, cell lysates containing an equal amount of proteins were incubated with anti-Flag agarose beads (Sigma-Aldrich), GFP-Trap agarose beads (Chromotek), or antibody-conjugated Protein A Magnetic beads (Merck) at 4 °C for 2 h. The beads were washed with lysis buffer...
Chapter Seven - Affinity Pull-Down of Proteins Using Anti-FLAG M2 Agarose Beads 热度: Anti-Digoxigenin-AP, Fab fragments - Sigma-Aldrich 热度: Sigma_Flag-M2 beads 热度: MonoclonalANTI-FLAG®BioM2−Biotin,CloneM2producedinmouse,purifiedimmunoglobulin ...
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crosslinked4%agarosebeads. EZviewRedANTI-FLAGM2AffinityGelfunctionsto bindFLAG ® fusionproteinsfromcelllysatesandother biologicalsamplesinthesamemannerasthe standardnon-coloredANTI-FLAGM2Monoclonal AntibodyAgaroseAffinityGel(CatalogNumber A2220).FLAGfusionproteins,boundtotheANTI- FLAGAffinityGel,arerecoveredby...
After centrifugation at 14,000 rpm for 10 min at 4 °C, the supernatants were incubated with Flag-agarose beads (A2220, Sigma-Aldrich) at 4 °C overnight. After that, the beads were washed five times with NETN buffer and the immunoprecipitates were collected and boiled in 50 μL of ...
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25 ng of N-terminal DYKDDDDK-tagged GFP protein was spiked into A549 lysate and captured using Pierce Anti-DYKDDDDK Magnetic Agarose or Sigma Anti-FLAG™ M2 Magnetic Beads using the KingFisher Flex Purification System. Tagged proteins were eluted with IgG Elution Buffer and analyzed by western bl...
25 ng of N-terminal DYKDDDDK-tagged GFP protein was spiked into A549 lysate and captured using Pierce Anti-DYKDDDDK Magnetic Agarose or Sigma Anti-FLAG™ M2 Magnetic Beads using the KingFisher Flex Purification System. Tagged proteins were eluted with IgG Elution Buffer and analyzed by western bl...
The desired protein was precipitated with appropriate antibodies conjugated directly to Sepharose beads (such as Flag M2 beads, Sigma) or via protein A/G-conjugated Sepharose beads. About 2 mg total protein worth of cell lysates were incubated with the antibody-conjugated beads for 1 h at ...
The rest of the supernatant was incubated with 5 µL of pre-washed and pre-blocked Anti-FLAG® M2 Magnetic Beads (Sigma-Aldrich, M8823) (blocked with 3% BSA in PBS for 30 min at 4 °C with constant rotation) overnight at 4 °C with constant rotation. The next day the...