Pepsin Solution Preparation¶

Version: v3.2
Last modified: 2026-09-08

Required Reagents¶

Pepsin: Sigma-Aldrich P6887-250MG (250 mg) — product page · Product Information Sheet (P6887pis Rev 04/22)
RNase free Water
12 M concentrated hydrochloric acid

Product Information¶

From the P6887 Product Information Sheet.¹

Property	Value
Form	Lyophilized powder
Specific activity	≥3,200 units/mg protein
Unit definition	One unit produces a ΔA₂₈₀ of 0.001 per min at pH 2.0, 37 °C, measured as TCA-soluble products using haemoglobin as substrate²
CAS / EC	9001-75-6 / 3.4.23.1 (Pepsin A)
Molecular mass	34,620 Da
pI	2.2–3.0
Solubility	10 mg/mL in deionized water; 4 mg/mL in cold 10 mM HCl
Inactivation	Irreversibly inactivated above pH 8; denatured at pH 8.5–11 at RT

units/mg protein, not U/μL

Earlier versions of this page listed the activity as >3200 U/μL. That was a unit error of three orders of magnitude, and it is the number every dose calculation starts from. The correct figure is ≥3,200 units/mg protein — note per mg protein, not per mg of powder, so the mass-based concentration is an upper bound on protein and the true U/mL can be lower.

Optimum pH is substrate-dependent¶

The datasheet does not give a single optimum, and the distinction matters for fixed tissue:¹

The optimal pH for the pepsin reaction is 1.5-2.5 […] Pepsin has an optimal activity with native proteins at pH ~1.0. However, with some denatured proteins, the optimal activity is in the range of pH 1.5-3.5.

Formaldehyde-crosslinked tissue protein is neither native nor fully denatured, so both 0.01 M HCl (pH ≈ 2) and 0.1 M HCl (pH ≈ 1) fall inside pepsin's working range. They are not equivalent, and the difference on fixed tissue has not been measured here.

Resolved in v3.0 — 0.1 M HCl throughout

This page said 0.01 M HCl from v2.0 while every protocol page said 0.1 M, and both have been used at the bench. The conflict is closed in favour of 0.1 M (pH ≈ 1) for every use:

It is what the padlock/RCA literature uses, without exception — Mignardi, Ke lab ×3, El-Heliebi, van den Heuvel, Bersani, Carow, Lawrence, Sankowski, Andersson, Kalhor, Roberts & Bassett. The 0.01 M value entered our tree from an FFPE smFISH protocol,³ a different lineage.
Acid-only permeabilization requires it. Where the protease is dropped entirely — Pre-treatment: FF v6.0, following SCRINSHOT and HybISS — the acid is the permeabilizing agent and 0.01 M is far too weak. One molarity across both routes removes a variable.

Runs made following this page before 2026-08-18 used pH ≈ 2 and are a comparison boundary against runs after it. Note that the direction and size of the difference on crosslinked tissue is not known — the datasheet's own optima are substrate-dependent, and no published head-to-head of the two molarities exists for in-situ work. If a sample that previously worked degrades after this change, the acid is the first thing to put back on the ladder.

Preparation Steps¶

1. Prepare 1 M HCl¶

Add 83 μL 12 M HCl to 917 μL RNase free Water (or 830 μL to 9.17 mL)
Mix well

2. Prepare 0.1 M HCl¶

Dilute 1 M HCl 10-fold: 100 μL 1 M HCl + 900 μL RNase free Water
Mix well. This one solution serves both the pepsin stock and the working solutions, and is also the acid used for acid-only permeabilization in Pre-treatment: FF.

3. Prepare 4 mg/mL pepsin storage solution¶

Weigh pepsin powder and dissolve to 4 mg/mL in cold 0.1 M HCl
Example: 4 mg pepsin + bring up to 1 mL with 0.1 M HCl
Prepare in small single-use aliquots and use fresh — see Storage and autolysis below

If it will not fully dissolve, drop the stock to 2 mg/mL

The datasheet quotes solubility as 4 mg/mL in cold 10 mM HCl and 10 mg/mL in water; it does not quote a figure for 0.1 M.¹ In practice the ISS literature routinely makes pepsin at 0.5–2 mg/mL in 0.1 M HCl, so a 2 mg/mL stock is a safe fallback — halve the stock and halve the dilution factor (1:20 for the 0.1 mg/mL working solution) to reach the same place.

4. Working solution: 0.1 mg/mL pepsin in 0.1 M HCl — the default¶

Dilute the 4 mg/mL storage solution 1:40 into 0.1 M HCl
Example: 25 μL storage solution + 975 μL 0.1 M HCl → 1 mL at 0.1 mg/mL
Prepare fresh before use

5. Stronger variant: 0.4 mg/mL pepsin in 0.1 M HCl¶

For ladder arms, for tissue that under-digests, and for reading older records that used it as the default.

Dilute the 4 mg/mL storage solution 1:10 into 0.1 M HCl
Example: 100 μL storage solution + 900 μL 0.1 M HCl → 1 mL at 0.4 mg/mL

Which concentration to use

0.1 mg/mL is the working default. It is what Pre-treatment: FFPE v4.0 runs, for 30 min at 37 °C. As of v4.0 the concentration and the time both match the four published FFPE padlock/RCA protocols that converge there — three run 30 min (Mignardi 2015, Bersani 2023, Jiang 2023), one runs 20 (Carow 2023). Between v3.0 and v4.0 only the concentration matched and the time was a third of theirs; runs from that window are a comparison boundary.

0.4 mg/mL was the default before v3.0 and is kept as §5 because older records and ladder arms refer to it. Change one variable at a time — concentration or time or acid, never two. If a section looks over-digested at 30 min, step the time down to 20 min; do not reach for the concentration.

Concentrations are stated in mg/mL, not %(w/v)

Older lab records use percentages. The mapping, for reading them: 0.04% (w/v) = 0.4 mg/mL, 0.01% = 0.1 mg/mL, 0.05% = 0.5 mg/mL, 0.4% = 4 mg/mL. Percentages are kept only where they are the natural unit — v/v dilutions such as PBS-Tween 0.05% or 70% ethanol.

What to write down, and in which units¶

The instruction stays %(w/v) / mg/mL — that is what you weigh and dilute, and it is exactly reproducible within one catalogue number. Do not restate the protocol in units.

The catalogue number and lot are what make mg/mL mean anything, so they are mandatory in the run log. P6887 is ≥3,200 U/mg protein; P7000 is ≥250 U/mg. At the same 0.4 mg/mL those differ ~13-fold in activity, so "0.4 mg/mL pepsin" without a P-number is not a reproducible dose.

The field's 0.1 mg/mL is a convergence in mass, not in activity

Of the four FFPE padlock/RCA protocols that land on 0.1 mg/mL, only one names a grade:

Paper	Product	Activity stated
Mignardi 2015	Sigma, CAS 9001-75-6 only — no catalogue number	—
Bersani 2023	Roche (different vendor)	—
Carow 2023	not stated	—
Jiang 2023	Sigma P7012	≥2,500 U/mg

So at 0.1 mg/mL:

Jiang, P7012: ≥250 U/mL
Ours, P6887 (≥3,200 U/mg): ≥320 U/mL — at least 1.28× Jiang

Both figures are floors, not points. A P6887 lot assaying at 4,000 U/mg would put 0.1 mg/mL at 400 U/mL, i.e. 1.6×. The overshoot is real but bounded, and it is smaller than the spread the field itself tolerates — those same papers run 20 min and 30 min, a 1.5× difference in time. That is the argument for adopting 0.1 mg/mL as-is rather than back-calculating a mass to match one paper's activity.

The fix that actually resolves it: pull the Certificate of Analysis for your lot. Sigma publishes lot-specific CoAs, which turn "≥3,200" into a measured number. Record that number, not the catalogue floor — it is the only way the U/mL in the table below means anything.

U/mL is for comparing with papers, not for specifying our protocol

Activity units are not portable between suppliers, and this is the reason not to make U the primary spec:

Unit	Assay conditions
Sigma unit (what P6887 is sold in)	ΔA₂₈₀ 0.001/min, pH 2.0, 37 °C, haemoglobin, TCA-soluble products
Anson unit (AU)	pH 5.5, 25 °C, 10 min, TCA-soluble products measured colorimetrically against a tyrosine equivalent
Milk-clotting / NF units	different substrate again

Different pH, different temperature, different substrate, different detection. There is no clean conversion between them, and published comparisons of standard pepsins give materially different activities depending on which method was used. A number in "U/mL" is meaningless unless the definition travels with it.

So: use U/mL only to place our condition against a published one, and always carry the definition with it. Everything in the table below is in Sigma units as defined above, computed from the P6887 spec — it is a comparison aid, not a dosing instruction.

Nominal	mg/mL	Sigma U/mL (≥3,200 U/mg protein)	at 2 min	at 10 min	at 30 min
0.4 mg/mL	0.4	≥1,280 U/mL	≈2,560 U·min/mL	≈12,800 U·min/mL	≈38,400 U·min/mL
0.1 mg/mL	0.1	≥320 U/mL	≈640 U·min/mL	≈3,200 U·min/mL	≈9,600 U·min/mL
Stock	4.0	≈12,800 U/mL	—	—	—

The bolded cell is the current FFPE v4.0 condition.

Two published reference points, and they do not agree with each other — which is the honest state of the field, not a transcription error:

The only pepsin titration read out on an in situ sequencing platform optimises at ~250 U/mL for 1–4 min depending on tissue,⁴ i.e. ~250–1,000 U·min/mL.
Published padlock/RCA protocols run higher: ≈ 1,250 U·min/mL on fresh frozen (Lawrence 2024 — 0.5 mg/mL P7012 in 0.1 M HCl, 37 °C, 1 min) and ≈ 7,500–9,600 on FFPE (Jiang 2023 and Mignardi 2015, both 0.1 mg/mL for 30 min).

Our FFPE condition sits with the second group by design. The retired fresh-frozen condition (0.4 mg/mL, 2 min ≈ 2,560) sat about 2× Lawrence — worth stating plainly because it was once suspected of over-digesting, and that suspicion was withdrawn on 2026-09-08; see Pre-treatment: FF v6.2. Titrate before committing a new sample type — the internal evidence review Permeabilization / Pepsin Digestion (lab site → Research) carries the comparison and a ready-to-run digestion ladder.

Storage and autolysis¶

Powder: −20 °C, as supplied.
Solutions: the datasheet's stability statement is for pH 4.4 — stable at −20 °C for about 2–3 months.¹ It says nothing about a stock held at pH 2.
A 4 mg/mL stock in acid sits at pepsin's activity optimum — pH ≈ 1 since v3.0, pH ≈ 2 before it, both inside the working range — which means it is a self-digesting solution: pepsin autolyses under exactly the conditions that make it active. Freeze single-use aliquots, and treat "how long has this aliquot been at 4 °C" as a variable worth logging.
This is not hypothetical. In a published in-situ optimization, solutions made from a higher-specific-activity pepsin were found to be "less consistent" and "not proportional in activity" to those from a lower-activity product, attributed to autolysis.⁴

What makes a pepsin dose reproducible

Not %(w/v) on its own, and not U/mL on its own either. Record all four:

Catalogue number — pins the specific activity and the unit definition
Lot — specific activity is a range, not a point (P6887 is 3,200–4,500 U/mg)
%(w/v) or mg/mL — the operational instruction, exactly reproducible within one catalogue number
Stock preparation date — an acidic stock autolyses, so mass ≠ activity after storage

Then, and only for comparing against a published condition, derive U/mL and state whose unit it is.

Notes¶

Prepare working solutions fresh; avoid extended storage after dilution
Use RNase free reagents for all operations
Pre-warm the working solution to the incubation temperature before applying, and record the measured temperature, not the setpoint

References¶

Changelog¶

v3.2 — 2026-09-08 — Follows Pre-treatment: FFPE v4.0, whose digestion time moved from 10 to 30 min. MINOR: no recipe on this page changed — both dilutions were already documented and neither concentration moved. What changed is which condition is named as current, and the reference material around it.

The 30-min column. The exposure table now carries 30 min alongside 2 and 10, so the current FFPE condition (0.1 mg/mL ≈ 9,600 U·min/mL) can be read off directly rather than multiplied out. v3.1 said the concentration matched the published FFPE protocols while the time did not; as of v4.0 both do, and that sentence is replaced rather than left to mislead.

The comparison paragraph is corrected. It previously set our fresh-frozen condition against the Xenium XTO titration alone (~250 U/mL, 1–4 min) and concluded ours was "several times that exposure" — true, but it silently compared a padlock/RCA protocol against an in-situ sequencing one and read as an indictment. Both reference points are now given with their lineage: XTO at ~250–1,000 U·min/mL, published padlock/RCA at ≈ 1,250 (fresh frozen, Lawrence 2024) and ≈ 7,500–9,600 (FFPE, Jiang 2023 / Mignardi 2015). Against its own lineage the retired 0.4 mg/mL / 2 min fresh-frozen condition is about 2× Lawrence, not an outlier — which is why the over-digestion suspicion it once carried was withdrawn in Pre-treatment: FF v6.2.

Two pre-existing errors fixed in passing. The autolysis note still described the 4 mg/mL stock as being in 0.01 M HCl, which v3.0 changed to 0.1 M; it now names both and the pH either side. And a sentence in the titration paragraph had been left garbled ("the lab-private evidence review (the internal evidence review …)") by an earlier find-and-replace.

v3.1 — 2026-08-18 — The working default moves to 0.1 mg/mL, following Pre-treatment: FFPE v3.0. MINOR: no recipe on this page changed — both dilutions were already documented — only which one is named as the default. The digestion time stays at 10 min, so the change is a clean fourfold reduction in exposure with one variable moved; the published protocols that use 0.1 mg/mL run it for 20–30 min, so expect to have to lengthen the time rather than raise the concentration if signal drops.
v3.0 — 2026-08-18 — All pepsin solutions are now made in 0.1 M HCl, and concentrations are stated in mg/mL rather than %(w/v). MAJOR: the acid moves 10× (pH ≈ 2 → ≈ 1), which changes both the enzyme's activity and the acid's own contribution, so runs either side are not directly comparable. No pepsin concentration changed — 0.04% and 0.4 mg/mL are the same thing.

The acid. This page carried 0.01 M from v2.0 while Pre-treatment: FF, Pre-treatment: FFPE, rca-ffpe.md and five lab records said 0.1 M; both have been run at the bench, which made "which acid" an uncontrolled variable depending on which page an operator followed. Resolved toward 0.1 M because that is what the entire padlock/RCA literature uses, and because the FF route now permeabilizes with acid alone, where 0.01 M cannot do the job. The stock is now made in 0.1 M as well, so there is a single acid in the workflow and no dilution arithmetic. Stated honestly: no published head-to-head of the two molarities exists for in-situ work, and the datasheet's optima are substrate-dependent, so the direction and size of the effect on crosslinked tissue is unknown. If a working sample type degrades after this, put the acid back on the ladder.

The units. %(w/v) is replaced by mg/mL across the protocol tree, matching how the rest of our reagents are specified and removing a conversion step at the bench. Mapping for older records: 0.04% = 0.4 mg/mL, 0.01% = 0.1 mg/mL, 0.05% = 0.5 mg/mL, 0.4% = 4 mg/mL. Percentages are retained only where they are the natural unit — v/v dilutions such as PBS-Tween 0.05% or 70% ethanol.

What makes a dose reproducible. Catalogue number and lot are now mandatory in the run log: P6887 is ≥3,200 U/mg protein against P7000's ≥250 U/mg, so the same mg/mL differs ~13-fold in activity between products. U/mL is documented as a comparison aid only, always carrying its definition — activity units are not portable between suppliers (Sigma's ΔA₂₈₀ / pH 2.0 / 37 °C / haemoglobin assay versus the Anson unit's pH 5.5 / 25 °C colorimetric assay, with no clean conversion).

Also corrected in this release: activity respecified from the erroneous >3200 U/μL to ≥3,200 units/mg protein with its unit definition, the P6887 Product Information Sheet linked, the substrate-dependent pH optima quoted as the datasheet actually gives them, the autolysis caveat added (the datasheet's stability figure is for pH 4.4, while our stock sits where the enzyme is active), and the contradictory "20 mg/mL storage solution" line removed from Notes. - v2.0 — 2025-09-09 — Current release: 4 mg/mL storage solution in 0.01 M HCl, diluted 1:10 to the 0.4 mg/mL working solution, with the optional 0.01% variant. Version numbering starts at 2.0 here; v1.0 predates this page and is not recorded.

Sigma-Aldrich. Pepsin from porcine gastric mucosa, lyophilized powder, ≥3,200 units/mg protein — Product Information Sheet P6887pis Rev 04/22. PDF · product page. Source of the specific activity, solubility, substrate-dependent pH optima, and the storage/stability statements above. ↩↩↩↩
Unit definition as stated on the Sigma-Aldrich P6887 product specification. The same definition (ΔA₂₈₀ 0.001/min, pH 2.0, 37 °C, haemoglobin, TCA-soluble products) is used in the INFOGEST pepsin assay — see A tool for predicting pH and temperature effects on porcine and human pepsin activity, Sci Rep 2026, which reports porcine pepsin's optimum at pH 2 / 37 °C across a pH 1–7 sweep. ↩
Annaratone L, et al. Quantification of HER2 and estrogen receptor heterogeneity in breast cancer by single-molecule RNA fluorescence in situ hybridization. Oncotarget 2017;8(11):18680–18698. DOI: 10.18632/oncotarget.15727. The FFPE pre-treatment our protocols descend from; specifies 0.025% pepsin in 10 mM HCl. ↩
Roberts K, Bassett AR. Optimisation of Xenium automated in situ sequencing for PAXgene-fixed tissue samples. bioRxiv 2025.02.11.637091. ↩↩

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