Human CD40 is a member of the tumor necrosis factor (TNF) receptor family and is present on all B cells except plasma cells. CD40 is also found on some epithelial cells, carcinomas and lymphoid dendritic cells. CD40 plays an important role in B cell activation and the interaction with its ligand CD154 (CD40 Ligand) is essential for isotype switching (1). CD40 muIg fusion protein is recognized by anti-human CD40 antibody and binds to human CD154. This binding can be blocked by anti-CD154 antibody. Molecular Structure: A soluble fusion protein consisting of the extracellular (193aa) domain of human CD40 fused to murine IgG2a Fc (232aa), with a predicted monomeric molecular weight of 45.6kD. Applications: Suitable for use in FLISA and Flow Cytometry. Other applications not tested. Functional Application: CD40-muIg fusion protein is recognized by anti-human CD40 (C2392-02E). This binding can be blocked by using anti-human CD154 (C2548-84W1). Recommended Dilutions: Flow Cytometry: 1:20 (10ug/ml). 80ul of diluted solution labels 5x10e5 cells. Optimal dilutions to be determined by the researcher. Storage and Stability: Store product at 4C in the dark. DO NOT FREEZE! Stable at 4C for 6 months after receipt as an undiluted liquid. Dilute required amount only prior to immediate use. Further dilutions can be made in assay buffer. Caution: PE conjugates are sensitive to light. For maximum recovery of product, centrifuge the original vial prior to removing the cap. Flow Cytometry Protocol: 1. Ficoll prepared human peripheral blood mononuclear cells were stimulated 6 hours in the presence of 10ng/ml PMA and 1uM Ionomicin to upregulate CD154 expression. 2. Five x 10e5 cells per tube were washed and incubated for 45 minutes on ice with 80ul of a 1:20 dilution (10ug/ml) of C2392-02L. 3. They were washed three times, fixed and analyzed by Flow Cytometry using a lymphoid gate. 4. A 29% sub-population of the cells stained positive with a mean shift of 1.41 log10 fluorescent units when compared to a negative control. 5. Binding was effectively blocked when cells were preincubated 10 minutes with 20ul of 0.5mg/ml anti-CD154 antibody (C2548-84W1).
Molecular Weight:
45.6
Purity:
Purified by Protein A affinity chromatography from tissue culture supernatant.
Form:
Supplied as a liquid in 50mM sodium phosphate pH 7.5, 500mM potassium chloride, 150mM sodium chloride, 0.2% BSA, 0.04% sodium azide, 15% glycerol. Labeled with R-Phycoerythrin (PE). A565/A280=4.0.
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