Anti human gamma chain hrp

Nicotiana benthamiana ΔXTFT plants were grown in a growth chamber at 22°C with a 16-h light/8-h dark photoperiod. All fusion proteins and antibodies or antibody fragments were transiently expressed via agro-infiltration. Total soluble proteins (TSP) and proteins secreted to the apoplastic fluid (AF) were collected 4–5 days post-infiltration (Kriechbaum et al., 2020 (link)). Expression of EPO-fusions was analyzed by immunoblotting of SDS–PAGE or native PAGE using mouse anti-EPO (1:5000, MAB2871, R&D Systems, Minneapolis, MN, United States), anti-human IgG-HRP (HC and LC, 1:10,000, W4031, Promega, Mannheim, Germany), anti-mouse IgG-HRP (1:10,000, Sigma-Aldrich, A0545), and anti-human kappa-chain-HRP (LC, 1:1000, Sigma-Aldrich, A7164) antibodies. EPO-Fc variants were purified from agroinfiltrated leaves (800 mg) with rProtein A or Protein G Sepharose^TM Fast Flow (GE Healthcare) and with KappaSelect^TM (GE Healthcare) for EPO-CL, according to the manufacturer’s instructions. Heavy chains of cetuximab (Castilho et al., 2015 (link)), anti-ebola 13F6 (Castilho et al., 2011a (link)) antibodies, and 2G12-ScFvFc (Loos et al., 2011 (link)) were cloned previously, and their expression was analyzed with anti-human gamma-chain-HRP (HC, 1:1000, Sigma-Aldrich, A7164). All purified proteins were stained with Coomassie Brilliant Blue.

Apoplastic and purified recombinant proteins were fractionated by 10% SDS‐PAGE under reducing and non‐reducing conditions and either stained with Coomassie brilliant blue (CBB, G‐250) or analysed by immunoblotting.
DL variants were analysed by Western blots using both anti‐human gamma chain HRP (1 : 5000, Sigma‐Aldrich, A8775) and anti‐human kappa chain HRP (1 : 5000, Sigma‐Aldrich, A7164). Detection was performed with Clarity™ Western enhanced chemiluminescence reagents (Bio‐Rad), and images were captured with a Fusion Solo S image system (Vilber Lourmat).