[Histonet] re question regarding pH in retrieval solutions

[John Kiernan]

In connection with the pH of boiling-hot solutions, I asked Google the questions: "How do you measure the pH of a boiling hot solution? Is there a standard buffer for hot solutions?"  The AI answer was far too long (4 or 5 screenfuls) for a Histonet reply, beginning with:
Measuring the pH of a boiling hot solution (near 100C) is difficult because temperature drastically changes both the solution's chemical properties and the sensor's physical response. You must use specialized high-temperature sensors  and account for the fact that "neutral" pH at 100C is approximately 6.14, not 7.00.
1. How to Measure Boiling Solutions.  High-Temperature Electrodes: Standard pH probes are usually rated only up to 60-80C. For boiling solutions, you must use a glass body electrode with high-temperature electrolyte and a specialized reference system designed to resist "poisoning" or drying out at high heat.  ....
If you ask the same question, you should get the same long answer, which has some references at the end. I didn't follow these up.
John Kiernan, London, Ontario, Canada.
= = =


________________________________
From: Gudrun Lang via Histonet <histonet at lists.utsouthwestern.edu>
Sent: Sunday, April 12, 2026 3:36 AM
To: 'Carl Hobbs' <carl.hobbs at kcl.ac.uk>
Cc: histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: Re: [Histonet] re question regarding pH in retrieval solutions

Hi Carl,
I am always slightly overhelmed by your answers with my humble school-English 😉.
We work with the Roche instruments and detectionsystems. They describe the high-pH AR-solution as "buffer on Tris-basis" and the low-pH AR-solution as "citrate-buffer". So I have to believe, that those reagens are actually buffers. And according to literature pH plays a role in the retrieval-process.
Maybe I need some lessons in chemistry about pH, dissoziation of H+ in buffers and water. Apparently even pure water changes pH with high temperatures and gets slighty acidic.
Boiling in hot water also does the AR-job to a certain degree. Boiling brakes short-range-bonds in proteins and leads to denaturation (and/or koagulation). Denaturation should also play an important part in AR. Boiling causes also the reversal of the binding of methylol-adducts from formaldehyd - and therefore frees the epitopes.
With the calcium-binding-theory I have troubles, because I don't see where the calcium comes from. There are some antigens (like E-Cadherin) that need calcium for stabilization of the tertiary-structure. But are there so many of them?

But why do high-pH and low-pH buffers work different, if the actual working pH is the same?
One idea is, that it is not the boiling-part but the cooling-part. How the proteins fold again during the cool-down would presumably depend on the pH-milieu.

best wishes
Gudrun


-----Ursprüngliche Nachricht-----
Von: Carl Hobbs via Histonet [mailto:histonet at lists.utsouthwestern.edu]
Gesendet: Samstag, 11. April 2026 20:07
An: histonet
Betreff: [Histonet] re question regarding pH in retrieval solutions (Gudrun Lang)

I hope I set my email to plain text...will find out!
To paraphrase The Excellent Leonard Cohen's song: Nobody knows
?
Chuckle

Citric acid HIER soln is not a buffer if only Citric acid is used
NaOH is used to adjust pH to 6
To make the Citrate a buffer, one would have to use Citric acid and sodium citrate
I use just Citric acid adjusted to pH6 using NaOH
TRIS is, as it is buffered using HCL to pH9
Do any buffers behave as buffers, at such high temperatures?
After Shi, Catoretti was The Man to nail it....imho
Imho, TRIS v Citra HIER rarely work equally well
One or the other  ( or neither ) works on Pwax sections
I have no XP of TRIS v Citra working equally with any antibody
I have not achieved ever, a better result incorporating EDTA into my TRIS pH9
So, for my lab: no "EDTA- sequestering of calcium bound to proteins" is effective
My basic understanding is that high heat induces a dipole moment in many proteins( twisting without fragmentation)  thus epitopes are "revealed", for a time
Need the high heat because the proteins are Formalin-fixed
( if I leave my HIER sections overnight before probing with primary antibody I have a sig loss- of antigenicity)
If I perform HIER on Formalin - fixed cryosections, I can obtain a similar immunoreactivity if I subject sections to 90C HIER....only after an extended HIER time
However, never as high a dilution factor for primary ab
I posted images on the excellent IHC site, several yrs ago....sadly no longer in existence

Well, that is my "rather long in the business" opinion, Gudrun
Discuss?
Best wishes
Carl



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