Source

Lipid A derived from E. coli R515 (Re) LPS

Concentration

1mg/ml (0.5mg/ml for 250µg size) stabilised in sterile, double-distilled water (ddWater), without any additives

TLRpure^TM

No detectable TLR4 independent activity: standardised potent TLR4-specific agonist

Purity

Ultrapure. No detectable DNA, RNA and protein traces.

Purification Method

R-type (mutant/rough) LPS was isolated by a phenol-chloroform-petroleum-ether method. Semi-purified LPS was subjected to further re-extraction cycles and ultracentrifugation steps, extensively electrodialysed to yield TLRpure^TM LPS, from which Lipid A was generated by mild acid hydrolysis.

Sterility

Filter method: according to Ph. Eur. 9.

Passed according to specification:

• No growth in Thioglycolate medium at 30-35°C after 14 days.

• No growth in Soybean Casein Digest Broth (TSB) at 20-25°C after 14 days.

Endotoxin Content

Bacterial Endotoxin Test (kinetic turbidimetric LAL method) according to Ph. Eur. 9.

Endotoxin Content: >5,000,000 [EU/ml].

Appearance

Colourless, clear, aqueous solution

Handling

Keep sterile. Prepare working dilutions from pre-warmed (~40°C) Lipid A stock solution just prior to use. Ready-to-use, sterile stock solution is cell culture-grade. No solubilisation required. Do not pre-dilute in buffer (e.g. PBS) as this will lead to precipitation of Lipid A. To yield a 100µg/ml (1,000-100x) stock solution add 100µl of Lipid A to 900µl endotoxin-free and sterile ddWater (Cat. No.: IAX-900-002) (not PBS) and mix well.

Activity

Optimal concentration is dependent upon cell type, species, desired activation and analysis:
0.1-1.0µg/ml in vitro and 5-15mg/kg in vivo in animal rodent models. Does not activate any TLR other than TLR4 as tested up to 1µg/ml in relevant cellular systems (mouse macrophages).