HCV NS5A protein expressed from Escherichia coli was purified using Invitrogen Ni-nitrilotriacetic acid (Ni-NTA) agarose beads according to the manufacturer’s instructions. Firstly, ProtoArray® Human Protein Microarray v5.0 (Invitrogen) was incubated with blocking buffer (50 mM HEPES [pH 7.5], 25% glycerol, 0.08% Triton X-100, 200 mM NaCl, 20 mM reduced glutathione, and 0.1 mM dithiothreitol [DTT]) for 1 h at 4°C. Next, purified NS5A protein diluted in probing buffer (phosphate-buffered saline [PBS] containing 0.1% Tween 20) was added to the protein microarray. Following incubation at 4°C for 1.5 h, the array was washed five times in ice-cold buffer and treated with Anti-V5-Alexa Fluor 647 antibody (Invitrogen) for 1.5 h at 4°C. The images were scanned using a PerkinElmer ScanArray Ex-press HT system and analyzed by the Invitrogen Prospector software (ver. 5.2).
Anti v5 alexa fluor 647 antibody
Manufactured by Thermo Fisher Scientific
The Anti-V5-Alexa Fluor 647 antibody is a fluorescently labeled antibody that recognizes the V5 epitope tag. It is designed for the detection and localization of proteins tagged with the V5 epitope in various applications.
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2 protocols using anti v5 alexa fluor 647 antibody
1
Purification and Microarray Analysis of HCV NS5A Protein
2
Cryosectioning and Immunofluorescence Imaging
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Tissues were collected into OCT medium and immediately frozen on dry ice and transferred into − 80 °C where stored until analysis. Sections were cut on cryotome machine, hydrated with PBS, fixed in 4% formaldehyde, and blocked with PBS + 1% FBS for 1 h. 100 ng of MBA414 or 1 µg of anti-PD-1 antibody-FITC (Novus) were left overnight at 4 °C. Anti-V5 Alexa Fluor 647 antibody (Invitrogen) was used next day for 1 h. Mounting medium with DAPI (Merck) was used for final preparation of specimen for microscopy. Microscope Leica connected with camera was used for observation of tissues, software was used for preparation of images. Co-localization was quantified by ImageJ software (LOCI, University of Wisconsin) supplemented with plugin JACoP (Fabrice P. Cordelières, Bordeaux Imaging Center (France). Fabrice.Cordelieres@gmail.com, Susanne Bolte, IFR 83, Paris (France). Susanne.Bolte@upmc.fr).
Additional part of methodology is provided in Additional file. In this study, selection criteria for experimental analysis of MBA variants is given as Additional file1 : Table S2.
Additional part of methodology is provided in Additional file. In this study, selection criteria for experimental analysis of MBA variants is given as Additional file
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