Choosing cytokine signalling antibodies that report activity rather than presence: why a stat3 antibody and a phospho stat3 antibody answer completely different questions and why a stat1 antibody has the same split, what a jak1 antibody and a tyk2 antibody report upstream and how a socs1 antibody and a socs3 antibody mark the brake being applied, where an irf1 antibody, an irf3 antibody and an irf4 antibody sit as interferon and lineage factors, what an ifnar2 antibody detects at the receptor, and how an il-11 antibody, an il22 antibody, an il-13 antibody, an il-5 antibody, an il-8 antibody with its il8 antibody and il 8 antibody spellings, an il23r antibody, a tl1a antibody, a baff antibody and an epo antibody each need measuring where the ligand goes rather than where it was made

Designing a signalling experiment

1. Buy phospho and total as a pair. The phosphorylated fraction only means something as a proportion of the total, and both have to come from the same sample. Buying only the phospho reagent produces a result nobody can normalise.
2. Put phosphatase inhibitors in the buffer first. Phosphorylation is removed in a cold lysate within minutes. The inhibitor has to be present before the cells meet the buffer, and the path from plate to denatured sample has to be short.
3. Run a stimulation time course. Signalling peaks and decays on a scale of minutes. A single timepoint chosen for convenience regularly lands after the peak and reports a negative from a positive experiment.
4. Include a stimulated and an inhibited control. A ligand that fires the pathway and an inhibitor that blocks it bracket the assay and show the reagent tracks the biology. This pair is worth more than any isotype control.
5. Measure ligands where they go. Secreted cytokines leave the cell, so a lysate is the wrong sample. Measure the supernatant or the circulation, and state which form of the ligand the assay detects.

Minutes decide the result

This is the class of experiment where handling dominates. The difference between a clean phosphorylation signal and nothing at all is frequently ninety seconds and a buffer made without inhibitors.

Write the lysis procedure into the protocol with timings, prepare the buffer fresh, and have everything ready before the stimulation starts. Laboratories that do this stop having unexplained negative signalling results.

Receptor, kinase, factor and brake

A cytokine pathway is a chain, and measuring one link tells you little about flux. Receptor level, kinase activation, factor phosphorylation and the negative regulators induced afterwards each report a different part of the response over a different timescale.

Choosing two links from different points, and a late brake, gives a far more defensible picture than a deeper measurement of one.

Common questions

Why is my phospho signal absent?

Handling, in the great majority of cases: no phosphatase inhibitor, warm buffer, or too long between the plate and the denaturation step. Fix the lysis protocol before changing reagents.

Do I need the total reagent too?

Yes. The phosphorylated fraction is interpretable only relative to how much protein is present, and treatments frequently change both. Reporting phospho alone is uninterpretable.

Should cytokines be measured in lysate?

No. Secreted mediators are measured in the supernatant or in circulation. A lysate reports what has been made and not yet released, which is a different and usually less useful quantity.

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