What are the possible methods to prevent the digestion of antibodies (mainly Polyclonal) by proteases during affinity chromatography?

I read some papers about doing modifications to the anitbodies:

using unnatural amino Acids using mPEG But, are there better options, more commercially applicable?

I am trying to find a way to purify hyaluronidase from hepatopancreas of red king crab (using polyclonal antibodies). In this case, inhibitors like EDTA, Benzamidine, PMSF don't prevent proteolysis. These are the Hepatopancreas proteases: (Collagenolytic Serine Proteinase PC (PC—Paralithodes Camtschaticus), Collagenolytic Serine Proteinase PC 2, Trypsin-Like Proteinase A, Chymotrypsin-Like Proteinase C, Aminopeptidase PC, Carboxypeptidase PC, Trypsin PC, Elastase, Cathepsin L, Metalloproteinase)

  • $\begingroup$ You need more protease inhibitors, and likely higher concentrations of them too, given that you're working with extracts that are just chock full of proteases. Inhibitor cocktails like this one are available from many companies. Read the specific literature of work relating to this organ or just experiment empirically to determine optimal concentrations. $\endgroup$
    – MattDMo
    Feb 18 at 16:52
  • $\begingroup$ @MattDMo Thank you, but could there be other methods to isolate the anitbodies from proteases? During/before affinity chromatography? $\endgroup$
    – Alpha
    Feb 19 at 4:41
  • $\begingroup$ Protease inhibitors are really the tried and true method of protecting proteins, both the ones of interest in your sample as well as those complexed to the column beads. The problem with trying to modify the immunoaffinity antibodies is that you could very easily end up altering, damaging, or completely destroying their ability to bind to their substrate. That won't happen using inhibitors. I guess I should back up a bit - are you seeing damage to your antibodies, and is there a particular reason you can't/don't want to use inhibitors? $\endgroup$
    – MattDMo
    Feb 19 at 18:23
  • $\begingroup$ @MattDMo Inhibitors will be added anyway, but I was thinking of other possible ways to reduce protease activity from the crude extract before starting chromatography. Because in this case, relying on inhibitors won't be very effective, they don't work on all the proteases of hepatopancreas. $\endgroup$
    – Alpha
    Feb 20 at 5:02
  • $\begingroup$ The product I linked to (I have no affiliation with the company) has a number of other inhibitors than the ones you're currently using, targeting multiple different classes of proteases. That's what I suggest you look for - a "pan"-inhibitor cocktail designed for working with difficult extracts. As for other ways of reducing activity in the crude extract: you could try using detergents or chaotropic agents, but those will also interfere with antibody/antigen binding, and so I wouldn't recommend them. You could use heat to denature, but again you might interfere with Ab binding. $\endgroup$
    – MattDMo
    Feb 20 at 13:49


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