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Recombinant human CD14 protein
Background
CD14 acts as a co-receptor for the detection of bacterial lipopolysaccharide (LPS, endotoxin), which is a component of the outer membrane of Gram-negative bacteria [1]. CD14 is naturally expressed on the surface of certain immune cells, such as monocytes and macrophages, in a glycosylphosphatidylinositol (GPI) anchored form. CD14 also exists in plasma as a soluble form, lacking the GPI anchor (sCD14). Both membrane bound and soluble CD14 greatly enhance cellular sensitivity to LPS by promoting the transfer of LPS monomers from LPS-binding protein (LBP) to the TLR4/MD2 complex on the cell surface, which in turn triggers a signalling cascade that results in the production of inflammatory mediators. CD14 also enhances the sensitivity of cells to bacterial lipopeptides (BLP), by promoting the transfer of BLP to TLR2 [2].
Caithness Biotech recombinant CD14 comprises amino acids Thr 20 to Met 344 of the native human sequence, so lacks the GPI anchor lipid which is attached to the membrane-bound form at asparagine 345. Potential applications of sCD14 protein include use in bioassays to enhance cellular sensitivity to LPS, studies of the capacity of CD14 to transfer BLP, LPS and other lipids to various acceptors, and assays requiring monomeric display of LPS polysaccharides in a protein-bound form.
| Description | |
| Product | Recombinant human CD14 protein |
| Size / volume | 20 μg |
| Expression system | HEK-293 cells |
| Amino acids | Thr 20 to Met 344, accession number P08571 |
| Tags | C-terminal 6x His tag |
| Sequence graphic |  |
| Intended use | For laboratory research only, not for clinical or diagnostic use. |
| Specifications | |
| Format | Lyophilised from sterile PBS (pH 7.4) with trehalose as protectant and without additional carrier protein. |
| Purity | >95% by SDS PAGE |
| Molecular weight | Migrates at ~ 57 kDa (glycosylation present) |
| LPS content | <0.1 ng / μg (by HEK-293-TLR4 bioassay, relative to E. coli LPS standard) |
| Amino acid sequence | See Technical data sheet |
| Applications | ELISA / bioassay / SDS PAGE / binding studies / immunoassays |
| Pricing | |
| Catalogue number | CD14-1 |
| Price per unit | £100 per vial of 20 μg |
Figure 1: SDS PAGE analysis
10 μg of recombinant CD14 was separated by reducing SDS PAGE and visualised by Coomassie Blue staining. Caithness Biotech recombinant CD14 migrates at approximately 57 kDa due to glycosylation. See TDS for further information.
Figure 2: Validation of low levels of TLR4 stimulating contaminants
HEK-293 cells were transfected with NF-κB reporter and CD14 together with TLR4 and MD2, then treated with indicated concentrations of E. coli LPS, or the reconstituted protein at 1 μg/ml. NF-κB signalling was measured after overnight treatment by luminometry. See TDS for further information.
Figure 3: Validation of capacity of sCD14 to enhance sensitivity of TLR4 signalling complex to LPS
HEK-293 cells were transfected with NF-κB reporter and TLR4/MD2, but without a construct for expression of membrane-bound CD14. Cells were then treated with indicated concentrations of E. coli LPS in the presence or absence of reconstituted recombinant sCD14 at 100 ng/ml. All treatments were in DMEM lacking serum (0% FCS). NF-κB signalling was measured after overnight treatment by luminometry.See TDS for further information.
Product flyer
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Technical data sheet
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References
[1] Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249:1431-3 (1990)
[2] Nakata T, Yasuda M, Fujita M, Kataoka H, Kiura K, Sano H, Shibata K. CD14 directly binds to triacylated lipopeptides and facilitates recognition of the lipopeptides by the receptor complex of Toll-like receptors 2 and 1 without binding to the complex. Cell Microbiol 8:1899-909 (2006)