HSP70 antibody and chaperone reagents that report induction, not housekeeping

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.

An hspa1a antibody and the gene behind the inducible chaperone

HSPA1A is the inducible member of the family and is nearly identical to HSPA1B, so an hspa1a antibody raised on the shared sequence reports both and a clone claiming to separate them needs data. Because the protein is induced by stress, the harvest itself can raise it, so a handling protocol and an unstressed control belong in every comparison of levels.

An hsp27 antibody and the phosphorylation that sets its state

HSP27 works as a large oligomer that breaks into smaller units when phosphorylated, so an hsp27 antibody for total protein misses the state that matters and a phospho clone against a named serine is the measurement. The oligomers survive a non reducing gel, which is one way to show the shift. Total protein changes slowly while phosphorylation changes within minutes.

A grp94 antibody as the endoplasmic reticulum chaperone reference

GRP94 is an abundant resident of the endoplasmic reticulum, which makes a grp94 antibody a compartment marker for a fractionation as much as a subject in its own right. It is induced by stress in that compartment, so it is also a readout of that response, and those two uses pull in opposite directions: a marker should not move. State which role it is playing in the figure.

A bag3 antibody and the co-chaperone that directs disposal

BAG3 hands misfolded clients toward autophagy, so a bag3 antibody is read beside a chaperone and an autophagy marker rather than alone, and the readout of interest is usually its induction under proteotoxic stress. It forms complexes, so a native or crosslinked preparation is what shows the partner. Its own level is stress responsive, which makes the unstressed lane essential.

An hsp60 antibody and the mitochondrial folding machine

HSP60 is a mitochondrial matrix chaperone, so an hsp60 antibody doubles as a mitochondrial marker, and a cytoplasmic signal usually means the fractionation failed rather than that the protein moved. It runs near sixty kilodaltons where several abundant proteins sit, so a positive control and the datasheet's own blot matter. Import of the precursor means a slightly larger band can be real.

A pdi antibody and the oxidising environment it works in

Protein disulphide isomerase is abundant in the endoplasmic reticulum and is used both as a marker for that compartment and as a subject in redox work. A pdi antibody sees a family with several members, so the clone's specificity has to be stated. Because the enzyme forms mixed disulphides with clients, sample handling under reducing or non reducing conditions changes what the blot shows.

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 an ATF4 antibody or XBP1 antibody 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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