Hi, I'm trying to isolate my protein (Flag-tagged NOD2) in native conformation for BN-PAGE. I plan to IP the NOD2, I need to get the NOD2 off the beads/antibody without denaturing it. If I add DTT (let's say between 100-500mM) will this separate the protein and the antibody? Since IgG is held together by disulfide bonds, I'm thinking that the separation of the heavy and light chains would disrupt the protein-antibody interaction.
Has anyone tried this? Do you have any tips for an alternate method to isolate my protein in its native conformation? Thanks!
DTT might denature your protein. Perhaps try low-pH elution as it disrupts antibody-antigen binding. If your antibody is targeted to FLAG, you can use FLAG peptides to competitive bind to the antibodies so you can elute your protein more safely. Then do a dialysis trial to separate your protein from peptides.
I would assume you can also cleave the FLAG tag direcly if you are not using it later in your experiment.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA