MMP9 antibody and protease reagents for enzymes made inactive on purpose

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, and what an MMP-2 antibody cannot see

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.

A laminin antibody and the chain composition it has to name

Laminins are trimers assembled from several alpha, beta and gamma chains, so a laminin antibody sold under the family name is ambiguous and the useful datasheet names the chain. In tissue the stain is a basement membrane line, which is the pattern that says it worked. The chains are very large, so a low percentage gel and a long transfer are what make a band appear at all.

A loxl2 antibody and the crosslinking enzyme beside the matrix

LOXL2 crosslinks collagen and elastin and is secreted, so a loxl2 antibody may need conditioned medium rather than a cell lysate, and the proenzyme and processed forms give two bands. The lysyl oxidase family shares a catalytic domain, so specificity against the relatives has to be stated. Activity is an enzyme assay, and no antibody substitutes for it.

An fn1 antibody and the difference between soluble and fibrillar

Fibronectin exists as a soluble dimer and as an assembled fibrillar matrix, and the two answer different questions: an fn1 antibody on a lysate reports synthesis while a section or a deoxycholate insoluble fraction reports matrix. Splice variants are the point in some work, so the epitope has to lie in the region of interest. The protein is large and the gel has to suit it.

A cthrc1 antibody and a secreted marker of fibrosis

CTHRC1 is secreted and is read as a marker of an activated fibroblast state, so a cthrc1 antibody often reports medium as well as cells, and a comparison needs the medium volume and the collection time stated. It is glycosylated, so the band is broad, and the cell types expected to make it are the internal control for a tissue stain.

An adam17 antibody and the shedding it performs

ADAM17 sheds many surface proteins, which means it is usually the explanation for a falling surface signal elsewhere in an experiment rather than the subject. An adam17 antibody shows the proenzyme and the mature form at different masses, and only the mature form is active, so the expected bands carry the interpretation. Activity is measured with a substrate assay and an inhibitor control.

An mmp14 antibody and the membrane bound protease

MMP14 sits in the membrane rather than being secreted, which is why it is read in a membrane fraction and why an mmp14 antibody is used with a location rather than a concentration in mind. It is processed from a proenzyme and is also inhibited by TIMPs, so protein level and activity diverge. A zymogram or a fluorogenic substrate is what reports activity.

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 over an MMP2 antibody blot?

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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