The Thermo Scientific Pierce LAL Chromogenic Endotoxin Quantitation Kit is an efficient, quantitative endpoint assay for the detection of gram-negative bacterial endotoxins. Bacterial endotoxin catalyzes the activation of a proenzyme in the modified Limulus Amebocyte Lysate (LAL). The activated proenzyme then catalyzes the splitting of p-Nitroaniline (pNA) from the colorless substrate, Ac-Ile-Glu-Ala-Arg-pNA; the activation rate is proportional to the sample endotoxin concentration. After stopping the reaction, the released pNA is photometrically measured at 405-410nm. The correlation between absorbance and endotoxin concentration is linear in the 0.1-1.0EU/mL range. The developed color intensity is proportional to the amount of endotoxin present in the sample and can be calculated using a standard curve.
Important Product Information
•Accurate pipetting is critical for maintaining consistent results. A repetitive pipettor can aid in normalizing volumes between samples. Ensure pipetting order and rate of reagent addition remain consistent from well to well and row to row.
•All materials (e.g., pipette tips, glass tubes, microcentrifuge tubes and disposable 96-well microplates) must be endotoxin-free.
•Adjust the sample pH to 6-8 using endotoxin-free 0.1M NaOH or 0.1M HCl. Avoid pH-electrode contamination of the sample by testing the pH of a small sample taken from the bulk sample.
•Store samples to be tested to stop all bacteriological activity. Store at 2-8ºC for <24 hours and -20°C for >24 hours.
•Maintaining the correct temperature is critical for reproducibility. Use a proper heating block at 37ºC±1ºC. Do not use a cabinet-style incubator to perform the assay.
•Endotoxin adheres to glass and plastic surfaces; before pipetting, vortex solutions to ensure the correct endotoxin concentrations are measured.
•Glass tubes are preferred for making standard stock solutions; however, polystyrene or polypropylene microcentrifuge tubes (1.5 mL) may also be used. When using microcentrifuge tubes, dedicate the bag of tubes for the assay and follow aseptic techniques.
•If the test sample endotoxin concentration is >1.0EU/mL, dilute the sample five-fold in endotoxin-free water. Re-test