Biotinylated LPS S. abortus equi (S-form) TLRpure Sterile Aqueous Solution Cat. No. #IAX-100-009B

£175.81

Description Features Resources

Source	Biotinylated Lipopolysaccharide (LPS) from S. abortus equi, S-type (smooth/wild-type) 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 as determined by a mouse macrophage cell culture cytokine secretion assay using TLR4 deficient versus wild-type cells: standardised potent TLR4-specific agonist
Purity	Ultrapure. No detectable DNA, RNA and protein traces.
Purification Method	S-type LPS was isolated by a modified 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. TLRpure^TM LPS-Biotin was prepared using the biotin reagent biotinamidocaproate N-hydroxysuccinimide ester. Briefly, TLRpure^TM LPS at 10mg/ml in distilled water was mixed with biotin reagent in sodium bicarbonate buffer. The reaction mixture was stirred, dialysed extensively against distilled water in the dark, and sterile filtered.
Sterility	Filter method: certified 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) certified 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) LPS stock solution just prior to use. Ready-to-use, sterile stock solution is cell culture-grade. No solubilisation required. To yield a 100µg/ml (100x) stock solution add 100µl of LPS-Biotin to 900µl endotoxin-free and sterile ddWater (Cat. No.: IAX-900-002), 0.9% NaCl Solution (Cat. No.: IAX-900-003) or PBS (Cat. No.: IAX-900-001) and mix well.
Activity	Optimal concentration is dependent upon cell type, species, desired activation/staining and analysis: 1-10µg/ml. Does not activate any TLR other than TLR4 as tested up to 10µg/ml in relevant cellular systems (mouse macrophages). LPS binding to monocytes was evaluated by using LPS from S. abortus equi (S-form) Biotin (LPS Biotin) (as described in [1-3]). Briefly, for the kinetic studies, LPS Biotin (5µg/ml) was added to 100µl of heparinised whole human blood and incubated at 37°C in a 5% CO₂ atmosphere for different periods of time. A dose-response curve of LPS Biotin was performed with increasing concentrations of LPS Biotin and an incubation period of 20 min. Samples were incubated with CD14-FITC and HLA-DR PE monoclonal antibodies and with streptavidin-APC for 15 min in the dark, at room temperature. Monocytes were identified with the use of CD14-FITC and side scatter, and LPS Biotin positively stained monocytes with the use of streptavidin-APC. Shipping Ambient Storage 2-8°C (short-term storage). Prepare aliquots and store between -15 and -25°C (shelf-life 2 years). Avoid freeze/thaw cycles.

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