What is the best way to permanently deactivate or denature monoclonal antibodies? is there a temperature and/or pH value that would guarantee unrecoverable denaturation?
It's difficult to answer - thermal denaturation of antibodies normally leads to irreversible aggregation and happens at 70-90 degrees for IgGs, but stability, unfolding and aggregation behavior can vary significantly between different antibodies. e.g.
Schaefer, J. V., and Plückthun, A. (2012). Transfer of engineered biophysical properties between different antibody formats and expression systems. Protein Eng. Sel. Des. 25, 485-506. (http://www.bioc.uzh.ch/plueckthun/index.php?pid=3-1-34000)
It's difficult to answer - thermal denaturation of antibodies normally leads to irreversible aggregation and happens at 70-90 degrees for IgGs, but stability, unfolding and aggregation behavior can vary significantly between different antibodies. e.g.
Schaefer, J. V., and Plückthun, A. (2012). Transfer of engineered biophysical properties between different antibody formats and expression systems. Protein Eng. Sel. Des. 25, 485-506. (http://www.bioc.uzh.ch/plueckthun/index.php?pid=3-1-34000)
Maybe you could just reduce and alkylate all the cysteine present. This would probably irreversibly denature all disulfide bonded proteins in your solution. You can find a basic protocol here: http://proteomicsresource.washington.edu/docs/UWPR_Protocols_Protein_Digestion_Protocols.pdf [see Reduction / Alkylation (DTT, IAA and UREA)].
Hi Yaron, To break the Ab into Fab and Fc domains you could use Papain to ezymatically alter the structure. The Fab and Fc will retain their binding abilities. If your goal is to remove the antibody binding ability from a solution that contains antibodies then you are better off passing the solution over a protein G or A or L column and remove the antibodies. I suggest this because the only true way to damage the Fab fragment binding ability is to linearize the protein with detergent, reduction and heat (Laemmlli buffer, 2-ME, and 95C for 5 minutes). It may be, however, that your intention is to test something else in your serum or solution and just want to block the antibody. In that way you could add an inhibitor at saturating levels to the extent that the Fab fragments are all occupied. These are just a few ideas but please give more info on your goals and perhaps this community can suggest the best approach.
Cheers,
Chris
Thanks for the replies! My purpose is to block the activity of antibodies that are immobilized on a silicon AFM cantilever. We shape AFM cantilevers into ultra-sharp nanoneedles and then immobilize antibodies on them, in order insert them into live cells and to detect (by measuring unbinding forces) intracellular cytoskeletal proteins.
Basically, I want to block the activity of those silicon surface-immobilized antibodies so that they do not bind intracellular proteins when inside the cell.
Perhaps digestion with Papain is a good way. I don`t need to retain their form, just to completely digest / denature / lyze them so that the antigen would not specifically bind to the nanoneedle.
I was looking for a simple way such as heating, but maybe lyzing or protein-G blocking is a better way?
Thanks for any advice!
Yaron
Just simple, break the disulfide bridges between the different chains by using mercaptoethanol or DTT (Dithiothreitol) or DTE (Dithioerythritol). To speed up the reaction, some recipes requires a boiling procedure. Time at normal temperature helps you also. First the IgM is destroyed (1h, 0,01% ME as I remember this well), later on also IgG (at double concentrations).
Residual ME can be bound by adding free Cystein.
Beware: ME is neurotoxic!!!
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