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I find a mAb targeting protein A, which has beed validated to work in wb/IHC/IP or antibody conjugating drugs. I'm wondering if this mAb could be used for blocking the protein A from binding with other proteins? This mAb is the only one antibody which could be bought without having to make it myself, so the answer is really important for me. Looking forward for your responses.

jinyu huang
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No. Not all monoclonal antibodies block binding of other proteins. This depends entirely on the antibody and its epitope though. Most monoclonal antibodies target a small epitope, somewhere between 7 and 11 amino acid residues in length. Where this epitope sits on the protein will alter whether the antibody is capable of blocking binding of another protein.

If the epitope is at an interaction site, then it is entirely possible that it will be a blocking antibody, but if it sits somewhere else, then it may or may not block depending on proximity and size of binding site of both the antibody and the interacting protein. In this second case non-direct blocking is known as steric hindrance or steric blocking and is due to molecular size.

bob1
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  • Thansks for your really helpful reply! I wonna take this mAb in a vivo experiment. So does this mean I have to verify the blocking effect between protein A and other Abs before using? If so, wether just taking a flow cytometry staining is enough to verify the blocking effect. Is there any other experiments I have to do? – jinyu huang Aug 20 '23 at 02:21
  • @jinyuhuang You can verify blocking using Flow and potentially by microscopy (e.g. fluorescence using tagged antibodies). You could also do it by native western (i.e. non-denaturing) for simulation of what might happen in-vivo. You do need appropriate controls in each case to ensure that the effect seen is due to blocking and not some off-target effect or secondary antibody effect for example. Look up "antibody validation" for more information about how to validate your antibody; don't assume, just because it is monoclonal, that it can't have off-target interactions. – bob1 Aug 20 '23 at 02:27
  • Thanks for your suggestion again! This mAb had been used for antibody conjugating drugs which was approved by FDA. So, I guess there should be a extremely small chance of off-target interactions. I would like to investigate literature about flow cytometry, microscopy , and native western experiments as you suggest. – jinyu huang Aug 20 '23 at 17:04