Detecting proteases that are made inactive on purpose: why an mmp9 antibody, an mmp2 antibody, the mmp-2 antibody spelling beside it and an mmp14 antibody have to distinguish the latent zymogen from the active enzyme and why a total blot overstates activity, what a timp1 antibody and a timp2 antibody report as the inhibitors that decide the balance, how an adam10 antibody, an adam12 antibody and an adam17 antibody report sheddases whose substrates leave the surface, where a loxl2 antibody, a ctgf antibody, a cthrc1 antibody and a periostin antibody sit in remodelling rather than degradation, what an ngal antibody, an lcn2 antibody and a lipocalin-2 antibody name as one target under three labels and why it travels in complex with a protease, how a dpp4 antibody and an enpp1 antibody behave as surface ectoenzymes, and where an ager antibody and a rage antibody are the same receptor under two names

Measuring proteases honestly

1. Resolve zymogen from active form. The two differ in size and both are visible on a well resolved gel. Quantifying the total band as activity is the most common error in this area and it is avoidable by looking at the right band.
2. Use a zymography or activity assay for activity. Gelatin zymography and fluorogenic substrate assays measure what the enzyme does. They should accompany any claim about activity, because a blot cannot make it.
3. Measure the inhibitor alongside. Net activity depends on the enzyme to inhibitor ratio, and treatments frequently change both. Reporting the protease alone leaves the interpretation open.
4. Account for complexes. Several of these proteins circulate bound to partners, which changes their apparent size and can mask epitopes. A band at an unexpected size may be a complex rather than an artefact.
5. Measure sheddase substrates in the medium. When a sheddase acts, its substrate leaves the cell surface. Measuring the released fragment in the supernatant is a more direct readout of sheddase activity than blotting the enzyme.

Latency is the design of the system

These enzymes would be destructive if made active, so they are secreted latent and activated where and when they are needed. Every measurement decision follows from that: which band, which assay, which compartment.

A figure that shows both bands and names which was quantified is far more useful than one showing a single arrow.

The supernatant is often the sample

Secreted proteases, their inhibitors and shed substrates are all in the medium. A lysate reports synthesis, and the medium reports what was released, which is usually the biologically relevant quantity.

Collect conditioned medium under defined conditions, note the serum content, and include a medium only control, because serum contributes both proteases and inhibitors.

Common questions

Does more protease mean more degradation?

Not reliably. Most is present as inactive precursor and inhibitors are usually in excess. Net activity requires resolving the active form and measuring the inhibitors, or running an activity assay.

What does zymography add?

It reports enzymatic activity resolved by size, which a blot cannot. Its limitation is that the gel conditions can activate latent enzyme, so it is read as a comparison between lanes rather than as an absolute.

Why is my band at the wrong size?

Frequently a complex with an inhibitor or a carrier, which is real biology rather than an artefact. Run a reducing and a non reducing sample to see whether the shift is disulphide dependent.

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