Hek blue htlr4 cell line

Plain ELPs and the HN-ELPs were expressed in ClearColi BL21(DE3) electrocompetent cells (60810, Lucigen) following manufacturer’s protocol and purified using the inverse transition cycling (ITC). To determine the concentration, purified protein in PBS was mixed with the same volume of 6M Guanidine hydrochloride and assayed for absorbance using Nanodrop (Thermo Fisher Scientific, Waltham, MA). The final concentration is calculated using Beer Lambert’s law:
$$C_{ELP}=\frac{\left(A_{280}-A_{350}\right)}{\epsilon \times l}$$
where C_ELP is the molar concentration, A₂₈₀ and A₃₅₀ are absorbance at 280 and 350 nm respectively, l is the light path length in centimeters, and ε is an estimated molar extinction coefficient at 280 nm (1,285 M⁻¹cm⁻¹ for V96 or S96; 1,410 M⁻¹cm⁻¹ for HN-V96 or HN-S96). Protein purity and identity were further determined by SDS-PAGE gels (Lonza, Morristown, NJ) stained with GelCode Blue Safe Protein Stain (Thermo Fisher Scientific, Waltham, MA). Samples were probed by Western blot using an anti-humanin antibody.⁹ Protein molecular weight was confirmed by MALDI-TOF analysis through Proteomics and Metabolomics Facility at Colorado State University. The endotoxicity burden was also verified to be less than 6 EU/mL using the HEK-Blue hTLR4 cell line from InvivoGen (San Diego, CA)