List of tagged protein purification gel and elution peptide setsįor analysis of immunoprecipitated proteinsĬo-eluted antibody is not detected with HRP-labeled antibodies in Western blotting of immunoprecipitated proteins. List of anti-tag antibody-coated magnetic agarose beads List of anti-tag antibody-coated magnetic beads List of anti-tag antibody-coated agarose beads List of antibody-coated magnetic agarose beads Primary antibodies suitable for IP (excluding anti-tag antibodies) >The principle and method of polyacrylamide gel electrophoresis (SDS-PAGE) >The principle and method of Western blotting (WB)

Pull-down assay experiments using Tagged protein purification kits ※The antibodies are co-eluted, which should be taken into account when analyzing the data.Įlute the target protein in neutral pH using an elution peptide and avoid harsh conditions, such as acidic and alkaline solutions. Centrifuge, and use the supernatant for SDS-PAGE. Repeat 3-4 times.Īdd 50 µL of 2x SDS sample buffer containing 2-mercaptoethanol (2-ME) and heat for 5 minutes to elute the target protein from the beads. Remove the supernatant by aspiration without disturbing the beads. Pellet the agarose beads by centrifugation, and remove the supernatant by aspiration without disturbing the beads.Īdd 1 mL of ice-cold lysis buffer or washing buffer, mix, and centrifuge. Incubate at 4☌ for 1 hour-overnight on a rotating shaker. Incubate at 4☌ for 1 hour-overnight with shaking on a rotator.Īdd secondary antibody-coated (or protein A/G-coated) agarose beads. IP (with agarose beads)　※An example performed at MBLĪdd 500 µL of protein extract and 2-10 µg of the primary antibody to a 1.5-mL tube. Stable oligomeric complexes are formed because reaction between a polyclonal antibody and an antigen is multivalent. when the same antibody is used for both immunoprecipitation and Western blotting. If the binding affinity of an antibody is low, simultaneously using several high-specificity monoclonal antibodies will allow multivalent binding, resulting in stable antigen-antibody complexes. Special Considerations for Western Blots of Immunoprecipitates One. High-affinity monoclonal antibody (dissociation constant K d<10 -8 M) should be used because low affinity antibody may not form an antigen-antibody complex in solution.Įven if the affinity of individual antibody molecules is low, oligomeric antigen-antibody complexes are formed easily due to the multivalent binding. Background is low with an appropriate antibody because the antibody recognizes a single antigen.īackground may be high if some of the antibody molecules have a low specificity and bind to proteins other than the target protein.

Immunoprecipitation western blot