STAT3 antibody and cytokine signalling reagents that report activity, not presence

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.

Two links, not one: an IFNAR2 antibody for the receptor and a STAT1 antibody for the factor

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.

A phospho perk antibody and the residue it names

PERK activation is autophosphorylation, so a phospho perk antibody has to name the residue and be read against total PERK on the same membrane, and the protein's size means transfer conditions decide whether either appears. The response adapts within hours, so a single late time point can read as no stress response at all. A tunicamycin or thapsigargin treated lane is the control.

A tyk2 antibody and the family it belongs to

TYK2 runs close to JAK1 and JAK2 above one hundred kilodaltons and shares kinase domain sequence, so a tyk2 antibody needs cross reactivity data and a knockdown lane to make a band attributable. Activity is read as receptor and substrate phosphorylation after a cytokine stimulation, which is the control that shows the pathway rather than the protein was detected.

An src antibody and a kinase regulated in two directions

SRC is activated by dephosphorylation of an inhibitory tyrosine and by autophosphorylation of an activating one, so an src antibody for total protein says little and the phospho clones have to name their residues. The family is large and conserved, so specificity matters. It is membrane associated through a lipid modification, which makes the fraction part of the measurement.

An sgk1 antibody and a short lived kinase

SGK1 is transcriptionally induced and rapidly degraded, so an sgk1 antibody reports a condition rather than a constitutive protein and a proteasome inhibitor lane is often what makes the band visible. It shares substrate specificity with AKT, so a phospho-substrate readout cannot distinguish them and a selective inhibitor or a knockdown is what does.

A pka antibody in the same figure as its substrate

Where a cyclic AMP response is the subject, the informative pair is a phospho-substrate antibody against the kinase's recognition motif and a stimulation with a membrane permeable analogue. A pka antibody against a subunit confirms expression and supports a pulldown. The holoenzyme dissociates on activation, so the readout is substrate phosphorylation rather than a change in subunit level.

An lck antibody beside the receptor it serves

In a T cell signalling figure the kinase is read with the receptor chain it phosphorylates, so an lck antibody belongs beside a CD3 zeta phospho readout rather than alone, and the stimulation and its timing are what produce both. Its two regulatory phosphorylation sites have opposite effects, so a figure naming only 'phospho-LCK' has not said what it measured.

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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