Kinase abundance rarely changes on the timescale of a signalling experiment; what changes is phosphorylation. So every reagent in this class is bought as a pair, a total antibody for the denominator and a phospho clone that names its residue, with inhibitors in the lysis buffer and a stimulation time course that catches the peak rather than the adaptation.
An erk1/2 antibody and the two bands it reports
An erk1/2 antibody detects both isoforms, which run a few kilodaltons apart, and the activity readout is the doubly phosphorylated activation loop rather than either band's intensity. Both bands belong in the figure because the isoforms are not interchangeable in every tissue. The signal rises and falls within minutes, so the harvest time is part of the reagent choice.
A p-akt antibody and the residue the figure has to name
Serine 473 and threonine 308 are phosphorylated by different kinases and answer different questions, so a p-akt antibody has to state its residue and the total antibody on the same membrane turns a band into a ratio. Growth factor stimulation over a short series is the positive control, and a phosphatase inhibitor cocktail is what keeps the signal alive between the plate and the gel.
An s6k antibody and a p70 s6 kinase antibody are one target
The kinase is catalogued by its gene symbol and by its apparent mass, so an s6k antibody and a p70 s6 kinase antibody are often the same clone in two listings and searching one hides the other. Two isoforms arise from alternative starts. The informative measurement is phosphorylation of the hydrophobic motif, which a rapamycin treated lane should collapse.
A jnk antibody and a family with several isoforms
The stress activated kinases exist as isoforms of two masses from three genes, so a jnk antibody shows a pair of bands and a pan clone cannot attribute a change to one gene. Activity is read as the doubly phosphorylated loop or as phosphorylation of a substrate, and a stress stimulus with a stated duration is what makes the blot a pathway result.
A tak1 antibody and a kinase that needs its binding protein
TAK1 is active only with its binding partners, so a tak1 antibody reports the kinase while the complex is shown by immunoprecipitation and the activity by phosphorylation of a downstream kinase. It is itself phosphorylated on activation at a named residue, and an inflammatory stimulus with a short time course is the control that produces it.
A socs1 antibody and an inhibitor that appears then goes
SOCS1 is induced by the pathway it switches off and is then degraded, so a socs1 antibody reports a narrow window and a resting sample shows almost nothing, which is the expected result. A proteasome inhibitor lane holds it up. Because it is small and scarce, a positive control lysate from a stimulated line is what makes a faint band interpretable.
An sphk1 antibody and an enzyme read by its product
Sphingosine kinase 1 makes a signalling lipid, so an sphk1 antibody reports expression while the pathway is read by the lipid or by its receptors, and translocation to the membrane on activation means location carries information the level does not. The family relative shares sequence, so specificity against it is the line to read before a conclusion.
An fgfr antibody and the family the name hides
An fgfr antibody without a number is ambiguous, because the four receptors share sequence and their splice variants differ in ligand binding, so the useful datasheet names the receptor and the tested cross reactivity. Apparent mass moves with glycosylation, and the pathway is read downstream through phosphorylated ERK and FRS2 rather than through the receptor's level.
Questions people ask about tyk2 antibody
Why is a total kinase blot not enough?
Abundance rarely changes on a signalling timescale. The phosphorylation does, so the pair is the measurement.
What has to be in the lysis buffer?
Phosphatase inhibitors, added before the cells are scraped, or the signal is lost on the way to the gel.
Why a time course rather than one sample?
Most of these responses peak in minutes and adapt, so a late sample can read as no response.