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What bacterial endotoxin is, why it matters in laboratory work, how the Limulus amebocyte lysate assay measures it, and how to read an EU/mg figure.
Endotoxin is lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria. It is released when those cells die and lyse, which means it can be present even in material that is sterile.
It is thermally stable in a way that most biological contaminants are not. Autoclaving kills bacteria but does not reliably destroy endotoxin, so sterilisation and depyrogenation are separate problems.
Endotoxin is biologically active at extremely low concentrations and triggers strong responses in immune and endothelial cell lines. In cell culture work it is a classic source of results that cannot be reproduced.
If an assay involving immune-competent cells produces an unexpectedly strong response, endotoxin contamination in one of the reagents is worth ruling out before the result is interpreted.
The Limulus amebocyte lysate assay uses a clotting cascade from horseshoe crab blood cells that is triggered by endotoxin. The three common formats are gel-clot, which gives a pass/fail at a threshold; turbidimetric, which measures the rate of clouding; and chromogenic, which measures colour development from a synthetic substrate.
Chromogenic is the most quantitative and is what produces a numeric EU/mg figure.
Results are reported in endotoxin units per milligram of peptide. One endotoxin unit is approximately 0.1 to 0.2 nanograms of reference lipopolysaccharide.
Below 0.5 EU/mg is a normal specification for research-grade peptide. Lower figures are achievable but the cost of depyrogenated processing rises quickly, and for most in-vitro work below 0.5 is not the limiting variable.
Endotoxin is introduced by water, glassware and handling far more often than by the peptide synthesis itself. Depyrogenated glassware, endotoxin-free water and clean technique at reconstitution all matter.
A vial that leaves the supplier below specification can easily be pushed above it by reconstituting with ordinary laboratory water.
This guide is general laboratory reference material relating to the handling of research compounds. It is not medical, veterinary or clinical guidance, and it does not describe or imply any use in humans or animals. All PeptideSeed material is supplied for in-vitro laboratory research only — see the terms of supply.