HEK-blue™ CD40L responsive indicator cells were cultured with 0 to 2 μg/ml of hCD40L. Secreted embryonic alkaline phosphatase (SEAP) activity was assessed by measuring OD650.
The purity of recombinant hCD40L was determined by SDS-PAGE of 1.5 µg reduced (+) and non-reduced (-) recombinant hCD40L and staining overnight with Coomassie Blue.
Western blot analysis of extracts from THP-1 cells, untreated or treated with hCD40L for 15 minutes, using Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb #4668 (upper) and SAPK/JNK Antibody #9252 (lower).
Working concentration of hCD40L generally ranges from 1-100 ng/ml.
Recombinant human CD40L is supplied as lyophilized material that is very stable at -20°C. It is recommended to reconstitute with sterile water at a concentration of 0.1 mg/ml, which can be further diluted in aqueous solutions as needed. Addition of a carrier protein (0.1% HSA or BSA) is recommended for long term storage.
The bioactivity of recombinant hCD40L was determined by measuring the secreted embryonic alkaline phosphatase (SEAP) activity in HEK-blue™ CD40L responsive indicator cells. The ED50 of each lot is between 5-50 ng/ml.
Recombinant human CD40L was expressed in E. coli and is supplied in a lyophilized form. A greater than 95% purity was determined by SDS-PAGE. Endotoxin levels are less than or equal to 1 EU / 1 μg hCD40L.
CD40 Ligand (CD40L), a member of the TNF superfamily of ligands, is expressed as either a membrane-bound or soluble homotrimer (1). Both membrane-bound and soluble forms of CD40L have biological activity (1,2). CD40L is expressed primarily on activated T cells; however, mast cells, basophils, and NK cells may also express CD40L (1). CD40L functions as an important T cell co-stimulatory molecule and enhances T-dependent B cell responses (1,2). CD40L binds CD40, which is a receptor expressed on B cells, dendritic cells, and macrophages (1). Binding of CD40L to CD40 leads to the recruitment of TRAF proteins (TRAFs 2,3,5, and 6) resulting in the activation of the JNK and NF-κB pathways (1,2).
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