Kta avant 25

Fibers were purified from the virus as previously described for vaccinia virus (Jensen et al., 1996 (link)). Previous analysis of the fiber by 2D-gel coupled with MALDI-TOF MS (Boyer et al., 2011 (link)) revealed that three proteins (R135, L725, and L829) were associated with Mimivirus fibers. For anti-L725 antibodies, L725 protein fused with thioredoxin was expressed in Escherichia coli and purified using ÄKTA avant 25 (GE Healthcare, USA). Purified Mimivirus virions, fibers, and L725 protein were injected into mice to obtain anti-L725 polyclonal antibodies, as previously described (Boyer et al., 2011 (link)).

Analytical size-exclusion chromatographies to purify E3 : E2 complex or molecular mass estimations were carried out using a Superdex 75 10/300 GL column (GE Healthcare Lifesciences, Sweden) in an ÄKTA Avant 25 chromatography system (GE Healthcare Lifesciences, Sweden). The column was first calibrated with the gel filtration molecular mass marker kit (GE Healthcare Lifesciences, Sweden). Individual protein samples or mixtures were applied to the column pre-equilibrated with GF buffer [20 mM Na-HEPES (pH 7.7), 150 mM NaCl, and 0.5 mM DTT] and elution profiles were monitored by recording the absorbance at 280 nm.

Analytical hFcRn affinity chromatography was performed using an ÄKTA Avant 25 instrument (GE Healthcare), essentially as previously described^{58 (link),68 (link)}. Briefly, 50 µl WT hIgG1 and Fc-engineered variants (1 mg/ml) were injected and eluted by a linear pH gradient from pH 6.0 to 8.8 within 110 min using 20 mM MES sodium salt, 140 mM NaCl pH 5.5 and 20 mM Tris/HCl, 140 NaCl, pH 8.8 as eluents. To determine the elution pH at particular retention times, the pH was monitored by a pH detector (GE Healthcare). HSA variants were analysed using 2 mg/ml followed by a pH elution gradient from pH 5.5 to 8.8 within 110 min using the same buffers as above.

The protein component was filtered through a 0.45-μm filter membrane before purification. ÄKTA Avant 25 (GE Healthcare, WA, United States) equipped with HiLoad 26/600 Superdex 200 pg (GE Healthcare) was used for size-exclusion chromatography. The protein component was loaded at the rate of 5 ml/min, and 20 mm Tris, pH 5.0 was used for elution at the rate of 3 ml/min. The eluates corresponding to different signal peaks were collected. Amicon Ultra Centrifugal Filters of 10k MWCO (Merck, NJ, United States) were used for the protein concentration at 3000 g and 4°C until the volume was concentrated to 200 μl. SDS-PAGE was used for separating the protein concentrates using a NuPAGE 10% Bis-Tris Gel (Thermo Fisher Scientific, MA, United States). The protein bands were excised from the gel. The protein profiles provided by Sangon (Shanghai, China) were used for identifying the protein bands.

The isolation of RISC fractions from virus infected plants was performed as described previously with slight modification [12 (link)–14 (link)]. Briefly, 40 g TSWV-infected, TZSV-infected or mock-inoculated N. benthamiana leaf tissue was homogenized in liquid nitrogen and the powder was resuspended in 40 mL loading buffer (10 mM sodium phosphate, pH 6.8). The homogenate was filtered through two layers of gauze and subsequently centrifuge for 15 min twice at 14,000 ×g at 4°C. Supernatant (40 mL) was loaded onto a 25×3.6 cm² column packed with 50 mL Bio-Gel HT Hydroxyapatite (Bio-Rad, Hercules, CA). Chromatography separations were performed on ÄKTA avant 25 (GE Healthcare). After washing the column with 300 mL loading buffer, the bound proteins were eluted using a linear gradient of sodium phosphate buffer (pH 6.8) from 10–200 mM at a flow rate of 1.0 mL/min. Fractions were analyzed for the presence of active RISC by RNA cleavage assay and stored at -80°C. Active RISC fractions (0.5~1 mL) isolated from Hydroxyapatite column were further fractionated on Superdex 200 Increase 10/300 GL column (GE Hearlthcare, UK) pre-equilibrated with elution buffer (10 mM sodium phosphate, pH 6.8) at a flow rate of 0.5 mL/min. Every fraction (600 μL) was analyzed for RNA cleavage activity and active RISC fractions were stored at -80°C until further use.

High pH reversed-phase fractionation was performed on an ÄKTA Avant25 (General Electric) coupled to a refrigerated fraction collection. Purified peptides were separated on a reversed-phase column BHE 2.1 cm × 5 cm (Waters) at a flow rate of 0.2 ml/min at pH 10. The binary gradient started from 3% buffer B (90% ACN in 5 mM ammonium formate pH 10), followed by linear increases to the first 40% B within 30 min, to 60% B within 15 min, and finally to 85% B within 5 min. Each sample was fractionated into 24 fractions in 400 μl volume intervals. The fractions were dried in a vacuum-centrifuge and reconstituted in water with 2% ACN and 0.1% formic acid and concatenated in eight fractions.
Each fraction was injected into a nanoELUTE nano liquid chromatography system (Bruker Daltonics), peptides (200 ng of digest) were separated within 60 min at a flow rate of 400 nl/min on a reversed-phase column Aurora Series CSI (25 cm × 75 μm i.d. C18 1.6 μm) (IonOpticks, Australia) with 50°C. Mobile phases A and B were water and acetonitrile with 0.1 vol% formic acid, respectively. The %B was linearly increased from 2 to 17% within 37 min, followed by an increase to 25% B within 15 min and further to 35% within 8 min, followed by a washing step at 85% B and re-equilibration.

Aliquots were collected from the prefilled syringes and diluted in sterile PBS to the following concentrations: 5.6 mg/mL (aflibercept), 1.2 mg/mL (ranibizumab) and 3.5 mg/mL (bevacizumab). The experiments were performed using an ÄKTA avant 25 (GE Healthcare). Aflibercept and bevacizumab were run on a Superdex 200 Increase 10/300 GL column (GE Healthcare), whereas ranibizumab was run on a Superdex 75 Increase 10/300 GL column (GE Healthcare). For all samples, 77 μl was injected by means of an auto-sampler (Spark Holland B.V.).