NFkB antibody selection for a factor whose activation is a change of address

Designing the experiment

1. Fractionate or image, do not blot whole lysate. Translocation is the activation event. Nuclear and cytoplasmic fractions on the same blot, or imaging of nuclear intensity per cell, are the two readouts that report it. A whole cell lysate averages the answer away.
2. Use inhibitor degradation as the early readout. The inhibitor is destroyed within minutes of stimulation and resynthesised afterwards. Its disappearance is a fast, unambiguous activation signal and needs a tight time course to catch.
3. Validate the fractionation itself. A nuclear fraction contaminated with cytoplasm invalidates the whole experiment. Blot a known nuclear and a known cytoplasmic marker on every fractionation, and show them in the figure.
4. Expect oscillation and design around it. The pathway oscillates rather than switching once, so a single late timepoint can catch a trough. Sample early and often, and report the time course rather than a representative lane.
5. Control with a receptor level block. Blocking at the receptor, or using cells lacking it, distinguishes pathway activation from a general stress response that moves the factor for other reasons.

Location is the measurement for an NF-kB antibody

Every useful experiment in this pathway is about where a protein is or whether another one still exists. That makes fractionation quality and imaging quantification the technical crux, rather than antibody sensitivity.

Imaging has an advantage worth noting: it reports the distribution across cells, and this pathway frequently activates in a fraction of a population rather than uniformly. A blot would report that as a modest average change.

The brakes are part of the signal

Negative regulators are induced by the pathway itself, so their appearance is evidence that the pathway ran even after the factor has returned to the cytoplasm. They are a useful late readout when the early window has been missed.

Including one in the panel turns a snapshot into a record of what happened, which is often more robust than trying to catch the peak.

An ikba antibody and the degradation that is the signal

The inhibitor is degraded within minutes of pathway activation, so an ikba antibody reports the pathway by a band that disappears, and a lysate taken too late shows the protein back again after resynthesis. A short time course is therefore the experiment. A phospho specific clone against the serines that mark it for degradation catches the step before the protein is gone.

A tlr4 antibody and a receptor that is hard to stain

Surface levels of this receptor are low and much of the protein is intracellular, so a tlr4 antibody often reads weakly on unpermeabilised cells even in a responsive line. Species differences are large enough that a clone validated in mouse says little about human. Function is read through the downstream response rather than the receptor level, which is why a stimulation control belongs in the figure.

A sting antibody and the translocation that reports activity

STING moves from the endoplasmic reticulum toward the Golgi on activation and is then degraded, so a sting antibody reports activity through location and through disappearance rather than through abundance. That makes a compartment marker in the same image part of the measurement, and a phospho specific clone against the activation residue the cleaner readout on a blot.

An asc antibody and the speck that is the readout

Inflammasome activation is scored as a single bright cytoplasmic speck per cell, so an asc antibody is an imaging reagent with a counting rule rather than an intensity measurement. Crosslinked specks are insoluble, so they are lost by a standard lysate and are recovered from an insoluble fraction. The proportion of cells with a speck is the number that belongs in the figure.

An aim2 antibody and the sensor it detects

AIM2 senses cytosolic DNA and assembles with ASC, so an aim2 antibody is bought alongside that reagent and read in the same cells. Expression is restricted and interferon inducible, which means an unstimulated negative and a stimulated positive are the two controls that make a band interpretable. The protein is small enough that a high percentage gel resolves it better.

Common questions

Why does the total factor level not change with an NFkB p65 antibody or RelA antibody?

Because activation is release and relocalisation, not synthesis. The factor is present in the cytoplasm bound to its inhibitor before stimulation and in the nucleus after it, and a whole cell blot sees the same total either way.

What is the fastest reliable readout?

Disappearance of the inhibitor, which happens within minutes and is easy to see on a blot. It requires a time course fine enough to catch it before resynthesis.

How do I show a fractionation was clean?

Blot a nuclear marker and a cytoplasmic marker on the same membrane and include them in the figure. A translocation claim without those controls is not evidence.

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