360500 | Huzzah® KLA

Huzzah® KLA, Human Serum Albumin / Kdo2 Lipid A Complex


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Huzzah® KLA

Huzzah® KLA, Human Serum Albumin / Kdo2 Lipid A Complex

A new preparation of the saccharolipid glycan, Kdo2-Lipid A (KLA), is a nearly homogeneous Re lipopolysaccharide (LPS) substructure with endotoxin activity equal to that of native LPS. KLA is comparable to LPS and its activity is reduced by >103 in cells from TLR-4 deficient mice. The advantage of KLA over LPS is that it is a reproducible, defined natural product, and it can be detected by ESI/MS at the low concentrations used to stimulate animal cells. The purity of KLA should also facilitate the structural analysis of its complexes with signaling receptors, such as TLR-4/MD2.

New research* shows 1μg KLA induces

the same response as 10μg LPS.

Perform experiments at half the cost of using LPS! Avanti’s new HSA lipid delivery system, Huzzah™, improves the cellular delivery of KLA through its conjugation with human serum albumin (HSA), a physiologically relevant carrier protein. This conjugate system improves the solubility of KLA in aqueous buffer. The Huzzah™ lipid delivery system is supplied as a lyophilized conjugate which can be directly and easily reconstituted in water or a buffer of choice and is ideal for studying the effects of poorly soluble bioactive molecules. Solubility: Reconstitute lyophilized conjugate in 2 mL of water or other aqueous buffer. This produces a stock solution of 85 - 110 µg/mL KLA*. A short sonication may be necessary to ensure complete dissolution. If necessary, filter sterilize the reconstituted conjugate prior to use in cell culture applications. *Exact concentration will vary from batch to batch. Refer to label for exact product concentration upon reconstitution as determined by mass spectrometric analysis.

Molar Ratio: KLA:albumin (1:1) Albumin Biological Characterization: microbial free; endotoxin < 1 IU/mL Albumin Lipid Characterization: ~0.07g/L phospholipids; ~0.02 g/L sphingolipids; ~0.1 g/L fatty acids; and triglycerides and cholesterols below the limit of quantitation of 0.1 g/L and 0.05 g/L
Hygroscopic
No
Light Sensitive
No
Purity
>99%
Stability
1 Years
Storage Temperature
-20°C
Molecular Weight
0.000
Exact Mass
0.000

Huzzahprostaglandinscombi.gif
Sample Preparation Protocol - Dissolving of KLA and Huzzah KLA
(1) KLA was dissolved in sterile DPBS (Ca Mg free) at a concentration of 1 mg/ml.The solution was sonicated for 15-20 min to dissolve.
We made 20 µl aliquots and stored them at -20°C in a non-defrost freezer.
On the day of use, an aliquot was thawed and diluted 1:10 (100 µg/ml) in DPBS (Ca Mg free) before adding to the cells.
(2) Huzzah KLA:
2.0 ml of sterile DPBS (Ca Mg free) was added to the stock vial (100 µg/ml final).
The vial was vortexed and then sonicated for 10-15 min.
20 µl aliquots were frozen at -20°C.

*The first experiment with Huzzah KLA was done with freshly made solution that had not been frozen.

Experimental Protocol
(a) Plated 1x10^6 RAW264.7 cells per 100 mm dish in triplicate for each treatment (DMEM +10% FBS) and incubated overnight at 37°C.
(b) Replaced with 8 ml SF-DMEM containing PBS or KLA or Huzzah KLA at the designated concentrations.
(c) Incubated for 24 hrs at 37°C.
(d) Collected 4 ml for extraction into 8 ml ice-cold ethyl acetate + 0.5% acetic acid containing PGE2-d4 and PGE2-G-d5 internal standards.
(e) Remove organic layer and dried under nitrogen gas.
(f) Resuspended samples in 100 µl methanol:water (1:1) and analyzed by LC-MS/MS for PGs and PG-Gs.