Detecting induced enzymes without over-reading the induction: why a cox2 antibody, and the cox-2 antibody spelling beside it, detects an enzyme that is nearly absent at rest and strongly induced, which makes the loading control and the exposure decisive, what an inos antibody reports on the same logic and why both are frequently over-interpreted from a single blot, how a cyp1a1 antibody reports induction of a drug metabolising enzyme and what microsomal preparation it needs, where a galectin 3 antibody and a galectin-3 antibody name one secreted lectin under two spellings that is measured in medium as well as in lysate, what a gm130 antibody marks as a Golgi structural protein used as a compartment reference, and what an ift88 antibody, an rpe65 antibody, a wfs1 antibody, a cd1a antibody and an fsp1 antibody each demand of the tissue they are used on

Measuring induced enzymes

1. Establish the linear range before quantifying. A target that goes from nearly absent to abundant will saturate the detection long before the untreated lane is visible. Run a dilution series and choose an exposure where both ends are on scale.
2. Normalise to total protein. Housekeeping proteins change under the stimuli that induce these enzymes. Total protein staining is the safer normalisation and should be shown alongside.
3. Prepare microsomes for membrane bound enzymes. Drug metabolising enzymes sit in the endoplasmic reticulum, and a whole cell lysate dilutes them. A microsomal preparation concentrates them and is the conventional sample.
4. Measure secreted proteins in the medium. A secreted lectin or cytokine leaves the cell, so a lysate reports what has been made and not released. Measure both and say which is being reported.
5. Use a compartment marker as a control, not a result. Structural organelle markers are references for fractionation and imaging. Treating a change in one as a biological finding usually reflects a change in the preparation.
6. Pair induction with activity where the claim is functional. An induced enzyme is not necessarily an active one. Where the conclusion is about activity, an activity assay or a product measurement is what supports it.

Fold change is an exposure artefact until proven otherwise

When the baseline is at or below detection, the calculated fold change is a property of the exposure rather than of the biology. Reporting a large number from such a blot is common and indefensible.

Report the range in which the measurement was linear, and if the control lane is genuinely below detection, say that rather than dividing by it.

Induction and activity are separate claims

Protein appearing is not the same as flux through the enzyme, which depends on substrate, cofactors and post translational state. Both are measurable and they answer different questions.

Where the conclusion is about the pathway rather than the protein, add a product or activity measurement. It is usually a simpler experiment than the blot.

Common questions

Why does my induced enzyme look infinitely induced?

Because the untreated lane is below detection at the exposure used. A dilution series and a longer exposure of the control, or a more sensitive substrate, put both ends on scale so a real fold change can be reported.

Do I need microsomes?

For membrane bound drug metabolising enzymes, yes. They are concentrated in a microsomal fraction and diluted in a whole cell lysate, which is why the conventional sample is the fraction.

Lysate or medium for a secreted protein?

Medium for what was released, lysate for what has been made and retained. They are different quantities and reporting one as the other is a common error.

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