Choosing signalling antibodies by the fragment the pathway produces: why a notch1 antibody, a notch2 antibody, a notch3 antibody or a generic notch antibody must be selected against the cleaved intracellular fragment rather than the receptor if activation is the question, what an apoe antibody, a bax antibody, a gr antibody, a cofilin antibody, a giantin antibody, an mycn antibody, an s1pr2 antibody, a tgfbi antibody, a b7-h3 antibody, an adrb2 antibody and an irf5 antibody each demand of fixation, extraction and controls, and why a datasheet that names only the protein is not enough for any of them

Choosing and validating

1. Decide which species of the protein answers your question. Full length receptor, cleaved fragment, phosphorylated form or a conformational state are different analytes. For cleavage activated pathways the fragment is the signal, and an antibody against the extracellular region will never report it.
2. Match extraction to the compartment. Membrane and nuclear proteins need extraction conditions that a standard lysis buffer does not provide, and a target that appears absent is frequently a target that stayed in the pellet. Fractionate when the compartment matters.
3. Choose fixation for the epitope, not for the tissue. Cross linking fixation masks many epitopes and requires retrieval; some epitopes survive only in alcohol fixation or in frozen sections. The application listed on the datasheet is a starting point rather than a guarantee.
4. Demand validation evidence in your application. A blot on an overexpression lysate does not validate immunohistochemistry. Look for knockout or knockdown validation, and where it is absent, run a reduction control yourself before building on the reagent.
5. Record clone and lot as part of the method. Two clones against the same target are different reagents and frequently give different distributions. A protocol that names only the target is a protocol that will not reproduce when the catalogue changes.
6. Include a biological control that moves the signal. A treatment or genotype expected to change the target is worth more than any isotype control. It shows the reagent tracks the biology rather than something constant.

One target, several analytes

Most disappointment with signalling antibodies comes from treating the gene name as the specification. The same gene produces a precursor, a mature form, cleavage products and modified variants, and a reagent is raised against a region that may be present in some of them and absent from others.

Read the immunogen region against the domain structure before buying. It takes ten minutes and it explains in advance most of what would otherwise be a month of troubleshooting.

Controls that actually control

An isotype control shows what a non specific antibody of that class does; it does not show that your antibody is specific. Reduction of the target, competition with the immunogen peptide, or a tissue known not to express it are the controls that carry weight.

Run one of them once per reagent, keep the image, and the reagent becomes something the laboratory can rely on rather than argue about.

Common questions

Why does an antibody work on a blot and not on a section?

Because fixation and epitope accessibility differ completely. Denatured, linearised protein on a membrane presents epitopes that a cross linked tissue section hides, and retrieval conditions have to be optimised for each reagent.

How do I detect an activated receptor?

By detecting the species that activation produces, usually a cleaved fragment or a phosphorylated form, with a reagent raised against the neoepitope. Detecting total receptor tells you about expression, not activity.

Is knockout validation necessary?

It is the strongest available evidence and increasingly expected. Where no knockout exists, a knockdown with a clear signal reduction, or a peptide competition, is an acceptable substitute if documented.

Why does my nuclear factor show only cytoplasmic staining?

Frequently an extraction problem rather than biology. Nuclear proteins need a buffer that opens the nucleus, and standard whole cell lysis leaves much of the target in the insoluble fraction.

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