Details
ACTIVITY TEST
Buffer Formulation 20mM Tris, 150mM NaCl, pH8.0, containing 1mM EDTA, 1mM DTT, 0.01% SKL, 5% Trehalose and Proclin300. Traits Freeze-dried powder Purity > 97% Isoelectric Point 8.5 Applications Cell culture; Activity Assays.
Figure. The binding activity of CD14 with LBP.
Cluster Of Differentiation 14 (CD14), also known as CD14, is a component of the innate immune system. CD14 acts as a co-receptor (along with the Toll-like receptor TLR 4 and MD-2) for the detection of bacterial lipopolysaccharide (LPS). CD14 can bind LPS only in the presence of lipopolysaccharide-binding protein (LBP). Although LPS is considered its main ligand, CD14 also recognizes other pathogen-associated molecular patterns such as lipoteichoic acid. Besides, Lipopolysaccharide Binding Protein (LBP) has been identified as an interactor of CD14, thus a binding ELISA assay was conducted to detect the interaction of recombinant human CD14 and recombinant human LBP. Briefly, CD14 were diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100μL were then transferred to LBP-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CD14 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50µL stop solution to the wells and read at 450nm immediately. The binding activity of CD14 and LBP was shown in Figure 1, and this effect was in a dose dependent manner.
CD14 also can enhance LPS-stimulated IL-8 secretion by THP-1 human acute monocytic leukemia cells. Therefore, THP1 cells were cultured in 24 well plates at a concentration of 106 cells/mL and stimulated by LPS (1μg/mL), then added of different concentrations of recombinant human CD14 (1μg/mL, 5μg/mL). After 24h later, the production of IL8 was determined in the supernatants by cytokine specific ELISA.
Result: When the concentration of CD14 was 5μg/mL, the secretion LPS-stimulated IL-8 can be significantly increased.
The standard curve was demonstrated as follows.
Figure. Effects of CD14 on IL8 production by THP1 cells.
USAGE
Reconstitute in 20mM Tris, 150mM NaCl (pH8.0) to a concentration of 0.1-1.0 mg/mL. Do not vortex.
STORAGE
Avoid repeated freeze/thaw cycles. Store at 2-8°C for one month. Aliquot and store at -80°C for 12 months.
STABILITY
The thermal stability is described by the loss rate. The loss rate was determined by accelerated thermal degradation test, that is, incubate the protein at 37°C for 48h, and no obvious degradation and precipitation were observed. The loss rate is less than 5% within the expiration date under appropriate storage condition.
Image
Figure. Gene Sequencing (Extract)
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Figure. SDS-PAGE
Figure. Western Blot
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