Choosing kinase antibodies in matched pairs: why an akt antibody and a p-akt antibody have to come from the same sample and why a pi3k antibody or a pten antibody upstream changes what either means, what an erk antibody, an erk1 antibody, an erk1/2 antibody, a phospho erk antibody, a mapk antibody and a jnk antibody report at successive points in the same cascade, where a gsk3b antibody, an s6k antibody, an s6k1 antibody, a p70 s6 kinase antibody, a ps6 antibody, a raptor antibody, an lkb1 antibody, an sgk1 antibody and a sik1 antibody sit downstream of growth signalling, how a pka antibody, a pkc antibody, a pkc delta antibody and a camkii antibody report second messenger routes instead, what an src antibody, an lck antibody, a shp2 antibody and a ptp1b antibody add about the kinase and phosphatase balance, and why a rhoa antibody and an arf1 antibody are activity assays rather than blots

Designing a kinase experiment

1. Buy phospho and total together, always. The two reagents must be raised for the same protein and used on the same sample, ideally the same membrane through fluorescent multiplexing. A phospho signal without its total is uninterpretable.
2. Name the site. Kinases carry several regulatory phosphorylation sites that report different things, including activation, inhibition and priming. A reagent described only as phospho is not a specification.
3. Put phosphatase inhibitors in first. Phosphorylation is stripped within minutes in a cold lysate. Inhibitors go into the buffer before the cells meet it, and the path from plate to denatured sample must be short and rehearsed.
4. Time course before single timepoint. Kinase activation peaks in minutes. One time course per cell type and stimulus establishes where the peak is, and every subsequent single timepoint is chosen from that curve.
5. Use an inhibitor as the specificity control. A selective inhibitor of the kinase should collapse the phospho signal without changing the total. That pair of lanes is stronger evidence than any datasheet.
6. Treat small GTPases as activity assays. Nucleotide bound state, not abundance, is what matters for these proteins, and it is measured by pull down with an effector domain rather than by a blot of total protein.

Pairs, sites and inhibitors

Three habits separate reliable kinase work from the rest: reagents bought in phospho and total pairs, sites named in the methods, and a selective inhibitor lane included as the specificity control. None of them is expensive and together they remove most of the criticism this class of figure attracts.

Fluorescent multiplexing makes the pair trivial, because both can be read from the same membrane without stripping.

Cascades are read at two points, not one

Measuring one node tells you little about flux through a cascade, because feedback and parallel inputs are everywhere. Choosing one proximal and one distal node, and a late negative regulator, describes the response far better.

Where the conclusion is about pathway activity rather than one protein, that three point picture is what makes it defensible.

Common questions

Why did my phospho signal disappear?

Handling, in most cases: missing phosphatase inhibitor, warm buffer, or too long between the dish and denaturation. Fix the lysis protocol before suspecting the reagent.

Which phosphorylation site should I measure?

The one whose regulation your hypothesis concerns, named explicitly. Different sites on the same kinase report activation, inhibition and priming, and reporting phospho without a site is not a result.

Can total kinase level substitute for activity?

No. Expression and activity are independent, and treatments commonly change one without the other. Where activity is the claim, measure phosphorylation at a defined site or run an activity assay.

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