Application

Coating a plate well (6 well plate) with this recombinant CD274 protein in a specific culture medium at 5-10 μg/well allows for use 1) as human T and B cells activation/differentiation studies or 2) as a potential biomarker protein for infectious diseases in vitro or 3) for auto-immuno disease diagnostic development.Use this procedure as a guideline to determine optimal coating conditions for the culture system of choice.1. Thaw CD274 and dilute to desired concentration using serum-free medium or PBS. The final solution should be sufficiently dilute so the volume added covers the surface evenly (5-10 μg/well, 6 well plate). 2. Add appropriate amount of diluted material to culture surface.3. Incubate at room temperature for approximately 1.5 hours.4. Aspirate remaining material.5. Rinse plates carefully with water and avoid scratching bottom surface of plates.6. Plates are ready for use. They may also be stored at 2-8 °C damp or air dried if sterility is maintained.

Preparation Note

The extracellular domain of recombinant human CD274 (19-238 aa, derived from BC074984) was constructed with codon optimization and expressed with a small T7-His-TEV cleavage site Tag (29aa) fusion at its N-terminal and expressed in E. coli as inclusion bodies. The final product was refolded using our unique “temperature shift inclusion body refolding” technology and chromatographically purified.

Sequence

MASMTGGQQMGRGHHHHHHGNLYFQG^GEFFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER