Leaves of C. quinoa inoculated with nitrite-treated virus solution were examined using a Multigel-21 imaging system (TOPBIO, New Taipei City, Taiwan) at day 7 post-inoculation under UV light with a wavelength of 365 nm. Fluorescence was visualized using a red filter (600 nm wavelength) to monitor local lesion formation. Fluorescent spots that were observed under UV light but did not cause necrotic lesions in C. quinoa leaves were picked and used as inocula and transferred to another C. quinoa leaf for isolation. Putative attenuated mutants were transferred to the systemic hosts N. benthamiana and tomato line SV-055 (Known-You Seed Co., Ltd., Kaohsiung, Taiwan) plants to verify their infectivity and pathogenicity. The test plants were kept in a plant growth chamber and a greenhouse, as mentioned above, for symptom observation for at least 3 weeks. Virus infection and titers were monitored with an indirect enzyme-linked immunosorbent assay (ELISA) using the antiserum against the PVMV CP (RAs PVMV-CP) [34 ] previously produced in our laboratory.
Multigel 21 imaging system
Manufactured by Top-Bio
The Multigel-21 imaging system is a multi-channel fluorescence imaging instrument designed for the detection and analysis of fluorescent signals in various applications, including gel electrophoresis, blotting, and microplate-based assays. The system provides high-quality images and accurate quantification of fluorescent signals.
Automatically generated - may contain errors
Lab products found in correlation
Ecl select western blotting detection reagent, by Cytiva (2 mentions)
0.45 m nitrocellulose membrane, by Cytiva (2 mentions)
Anti gfp antibody, by GeneTex (2 mentions)
Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
0.2 m nitrocellulose membrane, by Cytiva (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
4 protocols using multigel 21 imaging system
1
Screening Attenuated Virus Mutants in C. quinoa
2
Membrane Orientation of PalmGRET and GRET in sEVs
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To characterize membrane orientation of PalmGRET and GRET in sEVs, sEVs isolated from 0.22 µm filtered CM were serially diluted to the following concentrations: 31.25, 62.5, 125, 250, 500, 1000, and 2000 ng in 5 µL. Cell lysates were diluted to 2000 ng in 5 µL as positive controls. 5 µL of diluted sEVs, cell lysates, and double‐filtered PBS were dotted onto 0.45 µm nitrocellulose membranes (Amersham Bioscience) and blocked in 10% BSA supplemented PBS overnight at 4 °C. The membranes were immunoprobed with anti‐GFP antibody (GeneTex) diluted in 5% BSA in PBS or PBST overnight at 4 °C, wash three times in PBS or PBST for 30 min each, and incubated with HRP‐conjugated secondary antibodies for 1 h at room temperature followed by another 3 washes. The membranes were developed by chemiluminescence using ECL Select Western Blotting Detection Reagent (Amersham Bioscience) and MultiGel‐21 Imaging System (Topbio). Antibody dilutions and hosts are provided in Table S2 (Supporting Information).
3
Western Blot Analysis of Extracellular Vesicles
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Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS) with cOmplete protease inhibitor cocktail (Roche). Protein concentrations of EPs and cell lysates were determined using Pierce™ BCA Protein Assay Kit (Thermo Scientific). Protein lysates were separated using Bolt™ 4-12% Bis-Tris Plus Gels (Invitrogen) and transferred onto 0.2 µm nitrocellulose membranes (Amersham Bioscience, Little Chalfont, UK). The membranes were blocked with 5% BSA (Cyrusbioscience, Taiwan) in PBST (1X PBS, pH 7.4, with 0.1% Tween-20) for 1 h at room temperature and immunoprobed with primary antibodies overnight at 4 °C. The membranes were washed three times in PBST at room temperature, incubated with secondary antibodies for 1 h at room temperature followed by another three washes in PBST. For Western blots of purified sEV pellets from sucrose gradients, the membranes probed by HRP-conjugated secondary antibodies were developed by chemiluminescence using Trident Femto Western HRP Substrate kit (GeneTex) with MultiGel-21
Imaging System (Topbio, Taiwan). For western blots of protein knockdown, EV-293T-GRET/-PalmGRET subtypes, and EP-HCA1-WT/GRET/PalmGRET characterization experiments, the targeted proteins immunoprobed by IR-dye-conjugated secondary antibodies were imaged by ODYSSEY CLx (LI-COR Biosciences, Nebraska, USA). Antibody dilutions and hosts are provided in Table S2.
Imaging System (Topbio, Taiwan). For western blots of protein knockdown, EV-293T-GRET/-PalmGRET subtypes, and EP-HCA1-WT/GRET/PalmGRET characterization experiments, the targeted proteins immunoprobed by IR-dye-conjugated secondary antibodies were imaged by ODYSSEY CLx (LI-COR Biosciences, Nebraska, USA). Antibody dilutions and hosts are provided in Table S2.
4
Membrane Orientation of PalmGRET and GRET in sEVs
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To characterize membrane orientation of PalmGRET and GRET in sEVs, sEVs isolated from 0.22 µm-filtered CM were serially diluted to the following concentrations: 31.25 ng, 62.5 ng, 125 ng, 250 ng, 500 ng, 1,000 ng, 2,000 ng in 5 µL. Cell lysates were diluted to 2,000 ng in 5 µL as positive controls. 5 µL of diluted sEVs, cell lysates, and double-filtered PBS were dotted onto 0.45 µm nitrocellulose membranes (Amersham Bioscience) and blocked in 10% BSA supplemented PBS overnight at 4 °C. The membranes were immunoprobed with anti-GFP antibody (GeneTex) diluted in 5% BSA in PBS or PBST overnight at 4 °C, wash three times in PBS or PBST for 30 minute each, and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature followed by another 3 washes. The membranes were developed by chemiluminescence using ECL Select™ Western Blotting Detection Reagent (Amersham Bioscience) and MultiGel- 21Imaging System (Topbio). Antibody dilutions and hosts are provided in Table S2.
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