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Short answer: PMSF (phenylmethylsulfonyl fluoride; PubChem names it phenylmethanesulfonyl fluoride) is an irreversible serine protease inhibitor with a molecular weight of 174.19 g/mol. It is used at 0.1-1 mM in lysis buffers, but it breaks down quickly in water, so it is added fresh from a dry-solvent stock just before use.
This page is general reagent information for researchers and laboratory staff. It is not a protocol and not safety advice for a specific workplace; follow the supplier’s safety data sheet and your institution’s rules.
Definition and scope
What is PMSF, and what is its structure and molecular weight?
PMSF has the formula C7H7FO2S and a molecular weight of 174.19 (PubChem, CID 4784, read 2026-11-07; the same value is given on the Sigma-Aldrich P7626 product information sheet). Structurally it is a benzyl group attached to a sulfonyl fluoride. The sulfonyl fluoride is the reactive part: it reacts with the active-site serine of a serine hydrolase and covalently modifies it, which inactivates the enzyme permanently (Wikipedia, PMSF, read 2026-11-07, citing James 1978, Anal Biochem 86:574-579). In sample preparation it is one of several additives used to protect proteins from proteolysis, for example before SDS-PAGE or a western blot.
What inhibits serine proteases, and is PMSF enough?
PMSF is one inhibitor of serine proteases; related reagents include AEBSF, p-APMSF and HDSF (Wikipedia, read 2026-11-07). PMSF does not inactivate every serine protease. Palmitoyl-protein thioesterase is a documented example that PMSF does not inhibit (Das 2000, J Biol Chem 275:23847, as cited by Wikipedia). If a particular enzyme matters to your experiment, check that enzyme specifically rather than assuming PMSF covers it.
How stable is PMSF in water?
Not very. Sigma-Aldrich states that PMSF is very unstable in the presence of water, and gives these half-lives in aqueous solution at 25 C:
| pH | Half-life at 25 C |
|---|---|
| 7.0 | about 110 minutes |
| 7.5 | about 55 minutes |
| 8.0 | about 35 minutes |
Source: Sigma-Aldrich P7626 product information; the same three values appear in Wikipedia (both read 2026-11-07). Wikipedia adds that at 4 C and pH 8 PMSF is almost completely degraded after one day.
What this means in practice (editorial interpretation of those numbers): stability falls as pH rises, so a buffer at pH 8 loses half its PMSF about three times faster than one at pH 7. A lysis buffer that has been sitting with PMSF already in it is not equivalent to one made up fresh, and a long incubation may need the inhibitor topped up or a more stable alternative. Sigma’s instruction is to add PMSF to the aqueous buffer immediately before use.
PMSF stock solution, solubility and working concentration
How to make 100 mM PMSF solution
The concentration and solvent facts are as follows. Sigma-Aldrich says stock solutions may be prepared at 100 mM in anhydrous isopropanol or in anhydrous (100%, not 95%) ethanol. Because the molecular weight is 174.19, a 100 mM stock corresponds to about 17.4 mg per mL (calculated from the molecular weight). A 100 mM isopropanol stock stored at -20 C is described as stable for several months. The effective working concentration is 0.1-1 mM, which means a 100 mM stock is diluted roughly 1:1000 to 1:100 into the buffer.
Vendor pages differ. Other Sigma-Aldrich pages describe 200 mM stocks in dry solvent kept at 2-8 C for at least nine months, while the product information sheet cited above gives 100 mM and -20 C. Where sources disagree, use the figures on the datasheet of the lot you actually hold. Wikipedia cautions against DMSO as a solvent because it makes intact skin permeable to PMSF.
PMSF solubility
The practical solubility information in the sources is that PMSF is dissolved in anhydrous alcohol (ethanol or isopropanol) for stocks and then diluted into the aqueous buffer, where it is only briefly stable. The dry solvent matters because water degrades the compound.
Storage and handling hazards
PubChem (read 2026-11-07) lists the GHS signal word Danger, with H301 (toxic if swallowed) and H314 (causes severe skin burns and eye damage). Wikipedia reports an LD50 of 150-215 mg/kg, states that PMSF can inactivate acetylcholinesterase, and says it should be handled in a fume hood while wearing gloves. The current safety data sheet for your supplier’s product is the document that governs your workplace.
Where it matters for labs
A checklist built from the sourced facts above:
- Timing: add PMSF to the buffer immediately before use, not when the buffer is prepared (Sigma).
- Buffer pH: check the pH first; the half-life shortens from about 110 to about 35 minutes between pH 7.0 and 8.0 at 25 C (Sigma).
- Stock solvent: anhydrous isopropanol or 100% ethanol, not 95% ethanol (Sigma).
- Concentration: 0.1-1 mM final (Sigma).
- Coverage: PMSF is not a universal serine protease inhibitor (Wikipedia, citing Das 2000).
- Handling: H301 and H314 apply; avoid DMSO as a carrier (PubChem; Wikipedia).
Related methods pages: zymography detects protease activity in gels, and trypsinization uses a serine protease deliberately.
Common misunderstandings
- “Once it is in the buffer, it is protected.” The opposite: the inhibitor is the least stable component of the mix.
- “95% ethanol is fine.” Sigma specifies 100% ethanol, since the water in 95% ethanol degrades PMSF.
- “It inhibits all proteases.” It is a serine protease inhibitor that does not inactivate every serine protease; this page makes no claim about other protease classes.
- “PMSF syndrome.” PMSF here means the laboratory reagent. This page gives no medical information.
Frequently asked questions
What is PMSF?
Phenylmethylsulfonyl fluoride, an irreversible serine protease inhibitor (C7H7FO2S, 174.19 g/mol) used in protein extraction buffers.
How stable is PMSF in water?
Half-lives at 25 C are about 110, 55 and 35 minutes at pH 7.0, 7.5 and 8.0.
What inhibits serine proteases?
PMSF is one such inhibitor; AEBSF, p-APMSF and HDSF are related reagents. Not every serine protease is inactivated by PMSF.
How to make 100 mM PMSF solution?
Sigma-Aldrich gives 100 mM in anhydrous isopropanol or 100% ethanol (about 17.4 mg/mL), stored cold and diluted into the buffer just before use.
Sources
- Sigma-Aldrich, PMSF product information sheet P7626, https://www.sigmaaldrich.com/US/en/product/sigma/p7626 (read 2026-11-07).
- PubChem, compound CID 4784 (formula, molecular weight, GHS classification), https://pubchem.ncbi.nlm.nih.gov/compound/4784 (read 2026-11-07).
- Wikipedia, PMSF, https://en.wikipedia.org/wiki/PMSF (read 2026-11-07), citing James 1978, Anal Biochem 86(2):574-579, doi 10.1016/0003-2697(78)90784-4, and Das 2000, J Biol Chem 275:23847.
