Anti gfp hrp

Leaves collected from 6-week-old T₀ transgenic N. benthamiana plants were ground in liquid nitrogen and subsequently incubated for 30 min in an extraction buffer containing 150 mM NaCl, 50 mM Tris–HCl pH 8.0, 1.0 % IGEPAL^® CA-630 (Sigma) and one protease inhibitor cocktail tablet per 50 mL (Roche). The homogenate was centrifuged at 18,000 rpm for 20 min and the supernatant was incubated with GFP-trap_A^® beads (Chromotek) at 4 °C for 1–2 h. After incubation, GFP-beads were spinned down and washed six times with extraction buffer. Proteins were eluted from GFP-trap_A^® beads by boiling for 5 min, separated on an 8 % SDS-PAGE gel and electroblotted onto PVDF membrane (Bio-Rad). Accumulation of eGFP-tagged LecRK-I.9 and LecRK-IX.1 was detected by immunoblotting using anti-GFP-HRP (Miltenyi Biotec). Supersignal West Femto Chemiluminescent Substrate (Thermo Scientific) was used for signal development. Coomassie brilliant blue staining was used to indicate the amount of loading.

Proteins from purified EV-A71 virions and protein lysates were mixed with Laemmli buffer and separated by 10% SDS-PAGE. The proteins were transferred onto nitrocellulose membranes, which were blocked with 5% skimmed milk in 0.05% Tween-20 phosphate buffered saline (0.05% PBST) for 1 hour at room temperature. The pooled human serum samples were pre-treated additionally with RIDA RF-Absorbens (R-Biopharm AG, Germany) or DTT (Invitrogen, USA) for IgM- and IgG-specific antibody detection, respectively. The membrane was then incubated with 1:5000 diluted anti-GFP-HRP (Miltenyi Biotec, Germany), 1:300 diluted pooled human serum, 1:100 diluted Light Diagnostics EV-A71 monoclonal antibody 3323 (mAb 3323; Millipore, USA) or 1:1000 diluted EV-A71-specific mAb 979 (Millipore, USA) for 1 hour at room temperature, followed by secondary antibody incubation with the 1:5000 diluted HRP-conjugated polyclonal rabbit anti-human IgM (KPL, USA), 1:3000 diluted Amersham ECL human IgG, HRP-linked whole Ab from sheep (GE Healthcare, USA) or 1:5000 diluted HRP-conjugated goat anti-mouse (Gene Tex, USA) antibody. The immunoblot was developed with Clarity Western ECL Substrate (Bio-Rad, USA) and detected by chemiluminescence. The sizes of the protein bands were determined using the Precision Plus Protein WesternC Standard (Bio-Rad, USA).