Gfp b2 sc 9996 horseradish peroxidase hrp conjugated antibody

N. benthamiana leaves were harvested 2 days after infiltration, were frozen in liquid nitrogen, and were ground into powder with mortar and pestle. Total protein extraction was performed by reducing and denaturing proteins from the leaf powder 10 min at 95°C in Laemmli buffer (0.5 M Tris-HCl, pH 6.8, 10 mM dithiothreitol [DTT], 2% SDS, 20% glycerol) in order to avoid in vitro nonspecific degradation of the fusion proteins. Proteins were separated by 15–20% SDS-PAGE (Mini-PROTEAN® TGX™ Gels) and transferred onto a nitrocellulose membrane using Trans-Blot Turbo Transfer System (Bio-Rad, CA, USA). Transfert efficiency was assessed by Red Ponceau staining. GFP detection was performed in a single step using a GFP (B2): sc-9996 horseradish peroxidase (HRP)-conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein bands on immunoblots were detected using Clarity ECL Western Blot Substrate (Bio-Rad, CA, USA) using the manufacturer's protocol.

The Co-IP protocol was described previously [56 ]. IP was performed by affinity chromatography with GFP_Trap_A beads (Chromotek). Proteins were eluted from the beads by heating 10 minutes at 70 °C. Proteins were separated by SDS-PAGE and were transferred onto a polyvinylidene diflouride membrane using a Trans-Blot turbo transfer system (Bio-Rad, Munich). The membrane was blocked with 5% milk in Tris-buffered saline and Tween 20. GFP detection was performed in a single step by a GFP (B2):sc-9996 horseradish peroxidase (HRP)-conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA); RFP detection was performed with a rat anti-RFP 5F8 antibody (Chromotek, Munich) and an HRP-conjugated anti-rat antibody. Membrane imaging was carried out with an ImageQuant LAS 4000 luminescent imager (GE Healthcare Life Sciences, Piscataway, NJ). Instant Blue (Expedeon, Cambridge) staining of rubisco was used as a loading control.