Nanoorange protein quantification kit

The cytoplasmic and nuclear subfractions were isolated using a commercial kit (Nuclear/Cytosol Extraction Kit, BioVision Inc., USA) with some modification. The concentration of the isolated protein was assessed by fluorescence with the use of a NanoOrange™ Protein Quantification Kit (Invitrogen). The clearance of the isolated subfraction was confirmed by western blot analyses with an anti-GAPDH antibody for the cytoplasmic subfraction and anti-Lamin A/C for the nuclear subfraction. The isolated proteins were stored at −80°C for further analyses.

Lyophilized tissues from stems (2 mg), source leaves (3 mg), and dry seeds (1 mg) from at least five plants (n ≥5) were used for analysis of soluble proteins. Proteins were extracted following Zhang et al. (2015) (link). Diluted stem (1:10), source leaf (1:100), and seed (1:100) extracts were used to determine the protein amounts with the NanoOrange protein quantification kit according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA) and a Bio-Tek Synergy HT microplate reader (excitation, 480 nm; emission, 590 nm; Winooski, VA, USA). Sucrose levels in leaf phloem exudates were determined as described (Zhang et al., 2015 (link)). Total elemental N content was analyzed according to Sanders et al. (2009) (link).

The protein in the EPS was analyzed using NanoOrange Protein Quantification Kit (ThermoFischer) following the manufacturer protocol. Briefly, 30 μL of the sample was diluted in 1X NanoOrange working solution followed by incubation at 95°C for 10 min. The plates were then allowed to cool at room temperature for 20 min. Fluorescence measurements were carried out on a spectrophotometer for 1 sec using excitation/emission wavelengths of 485/590 nm.

The protein quantifications were performed using the Micro BCA™ Protein Assay (Thermo Fisher Scientific), the Qubit™ Protein assay kit (Thermo Fisher Scientific), and the NanoOrange™ Protein Quantification Kit (Thermo Fisher Scientific). A standard curve was prepared for both the microBCA and NanoOrange assays using Bovine Serum Albumin (BSA), with final concentrations (in μg/mL) of 100, 50, 40, 30, 20, 10, 5, 2, and 0. The OMV sample was diluted 1:3 dilution before use in the microBCA and NanoOrange assays per manufacturer’s instructions. The Qubit assay kit provides 3 standards for calibration, which were used followed by the BSA standards above run as samples to confirm the concentration of the BSA standards. The OMV samples were not diluted before running the assay on the Qubit™ 4 Fluorometer per manufacturer’s instructions.

sEV isolated from the CM were quantified using a NanoOrange Protein Quantification Kit (LIFE TECHNOLOGIES). Briefly, sEV were diluted 1:100 in the 1× NanoOrange working solution and incubated at 90 °C for 10 min. Then, the samples were cooled for at least 20 min and the fluorescence was read at 470ex–570em nm wavelength, using a Spectra Max Gemini plate fluorometer (MOLECULAR DEVICES, Sunnyvale, CA). To characterize sEV surface epitopes, 5 μg of sEV were analyzed using the MACSPlex Exosome Kit (MILTENYI BIOTEC) that allows the detection of 37 surface markers and two isotype controls. sEV were incubated with MACSPlex Exosome Capture Beads and with MACSPlex Exosome Detection Reagent CD9, CD63, and CD81 for 1 h at RT. Then, 1 ml of MACSPlex Buffer was added to each sEV containing tube, and left for 15 min at RT. The sEV bound to the Capture Beads were washed by centrifuging at 3000×g for 5 min, and then resuspended in 150μL of MACSPlex Buffer. Samples were analyzed with a FACS Canto II (BD BIOSCIENCES). Surface markers were calculated subtracting the median signal intensity of each bead of the control sample from the signal intensities of the respective beads incubated with sEV.