Choosing chaperone and stress response antibodies that report induction rather than housekeeping: why an hsp70 antibody has to distinguish the constitutive family member from the inducible one and what an hspa1a antibody names precisely, what an hsp27 antibody and an hsp60 antibody report in different compartments, where a grp94 antibody, a calreticulin antibody, a pdi antibody, an erp57 antibody and a kdel antibody mark the endoplasmic reticulum lumen and why an er marker antibody or endoplasmic reticulum antibody sold generically is a weaker choice, how an atf4 antibody, an atf6 antibody, an xbp1 antibody, a perk antibody and a phospho perk antibody read the unfolded protein response as a branched pathway, what a bag3 antibody adds about chaperone assisted degradation, and where a keap1 antibody, an nrf2 antibody, a bach1 antibody, a txnip antibody and a prohibitin antibody put oxidative stress on the same blot

Choosing stress response reagents

1. Name the family member, not the family. Chaperone families contain closely related members with different regulation. Check that the reagent's immunogen distinguishes the inducible member if induction is the question, and be sceptical of a pan family reagent used to claim a stress response.
2. Match the compartment. Cytosolic, mitochondrial and endoplasmic reticulum chaperones are different proteins with different markers. A generic organelle reagent is usually a poorer choice than a named resident protein, because you can then say what it marks.
3. Read the unfolded protein response as branches. The response has separate arms with separate readouts, and a stress can activate one and not another. Measuring one branch and reporting the response as a whole is a common overstatement.
4. Include a positive stress control. A known inducer on the same blot establishes the dynamic range and shows the reagent detects induction. Without it a modest change cannot be placed on any scale.
5. Watch loading controls under stress. Common loading references change under stress, which produces spurious normalisation. Total protein staining is safer for this class of experiment than any single housekeeping protein.

Induction is the measurement

Because these proteins are abundant at baseline, the meaningful number is a fold change under a defined stress rather than a presence or absence. That makes the loading control, the exposure and the linearity of the imaging system unusually important.

Run a dilution series once on your own lysates to establish where the blot is linear, and keep exposures inside it. Saturated bands make an induction look smaller than it was.

Relocalisation without a change in amount

Several stress proteins act by moving rather than by accumulating: to the nucleus, to a granule, to a membrane. A blot of whole cell lysate is blind to all of it.

Where relocalisation is the hypothesis, fractionate or image. It is a different experiment from the blot and it is the one that answers the question.

Common questions

Why is my chaperone signal huge and unchanging?

Almost certainly a reagent detecting the constitutive family member alongside the inducible one. Switch to a reagent whose immunogen distinguishes them, or accept that the blot cannot report induction.

Which branch of the unfolded protein response should I measure?

Whichever the hypothesis concerns, and preferably more than one, because stresses activate them differentially. Reporting one branch as the whole response is the most frequent overstatement in this area.

Are housekeeping loading controls safe here?

Often not, because stress changes their expression too. Use total protein staining for normalisation in stress experiments, and show the total protein image.

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