irdye 800cw goat anti mouse igg h l  (LI-COR)

Journal: Antiviral Research

Article Title: Development of antibody-based assays for high throughput discovery and mechanistic study of antiviral agents against yellow fever virus

doi: 10.1016/j.antiviral.2020.104907

Figure Lengend Snippet: Detection of YFV structural and non-structural proteins by Western blot assay . Vero cells were mock-infected or infected with YFV 17D at a MOI of 1 for 48 h ( A ). Huh-7.5 cells were mock-infected or infected with YFV Asibi at a MOI of 2.5 for 40 h ( B ). Proteins in the cell lysates were resolved on 4-12% NuPAGE Novex gels and transferred to nitrocellulose membranes. Shown in green are the bands detected after incubation with the indicated rabbit polyclonal primary antibodies against YFV proteins and the IRDye 800CW Goat anti-Rabbit IgG secondary antibody. β-actin, shown in red, is an internal control, and was detected by a mouse monoclonal primary antibody and the IRDye 680RD Goat anti-Mouse IgG secondary antibody. A pre-stained protein molecular weight marker, shown on the left, is used to estimate the size of proteins resolved by gel electrophoresis. The band expected for each of the YFV proteins is indicated on the right.

Article Snippet: IRDye® 800CW Goat anti-Mouse and anti-Rabbit antibodies, and IRDye 680RD Goat anti-Mouse IgG were purchased from LI-COR.

Techniques: Western Blot, Infection, Incubation, Staining, Molecular Weight, Marker, Nucleic Acid Electrophoresis

Journal: Antiviral Research

Article Title: Development of antibody-based assays for high throughput discovery and mechanistic study of antiviral agents against yellow fever virus

doi: 10.1016/j.antiviral.2020.104907

Figure Lengend Snippet: Establishment of two YFV NS4B antibody-based antiviral assays . Huh-7 cells were seeded on 96- well (A) or 384-well plates (B) and infected with YFV 17D at a MOI of 1, with either mock or indicated treatment, for 48 h. (A) In-cell western assay. YFV proteins were detected with indicated antibody against NS3, NS4B or pan-flavivirus monoclonal antibody 4G2 as primary antibody, with IRDye® 800CW goat anti-Rabbit or anti-Mouse as secondary antibodies. Cells were stained with DRAQ5 and Sapphire700. YFV protein expression (green) and cell viability (red) were visualized in LI-COR Odyssey. (B and C) High-content imaging assay. Representative images of NS4B and DAPI staining are shown as a function of doses of BDAA treatment (B). Quantitation of percentage of cells with positive NS4B signal and total fluorescence intensity were calculated based on multiple images taken from each well (n=6) and expressed as average ± standard deviation. * indicates P

Article Snippet: IRDye® 800CW Goat anti-Mouse and anti-Rabbit antibodies, and IRDye 680RD Goat anti-Mouse IgG were purchased from LI-COR.

Techniques: Infection, In-Cell ELISA, Staining, Expressing, Imaging, Quantitation Assay, Fluorescence, Standard Deviation

Journal: mAbs

Article Title: Novel monoclonal antibodies to the SERINC5 HIV-1 restriction factor detect endogenous and virion-associated SERINC5

doi: 10.1080/19420862.2020.1802187

Figure Lengend Snippet: Detection of SERINC5 in HEK293 cells transduced to overexpress SERINC5 isoform 1 (HEK293_S5.1). (a) HEK293_S5.1 cell extract was applied to two preparative wells of a 12% polyacrylamide gel, with a center lane containing labeled molecular weight standards. The MPX blotting system was applied to the blot to screen for mAb reactivity with (lanes 6–10) or without (lanes 1–5) competition with the matched immunizing peptides at 10 μg/mL. The mAbs were added at 0.5 μg/mL to lanes in the MPX as follows: 14C1-1 (1 and 6); 18B6-1 (2 and 7); 23E4-1 (3 and 8); 28E8-2 (4 and 9); anti-DDK plus a mix of all three peptides (5 and 10). The mAbs were detected using goat anti-mouse IgG-IRDye 800CW (green bands). The rabbit anti-clathrin heavy chain (HC) was added to all lanes and detected with goat anti-rabbit IgG-IRDye 680RD (red bands), as a protein loading control. (b). MFI of flow cytometry surface staining of HEK293_S5.1 with AF647 conjugated mAbs, as indicated; intracellular detection was performed using 28E8-2 (ICL4). Histograms indicate staining of the cells with (blue peaks) or without (red peaks) mAb pre-incubation with the respective SERINC5 peptides.

Article Snippet: Background wells contained only IRDye 800CW-secondary goat anti-mouse antibody.

Techniques: Labeling, Molecular Weight, Flow Cytometry, Staining, Incubation

Journal: The Journal of Cell Biology

Article Title: A toolbox of anti–mouse and anti–rabbit IgG secondary nanobodies

doi: 10.1083/jcb.201709115

Figure Lengend Snippet: Western blotting with infrared dye–labeled anti-IgG nanobodies. (a) A twofold dilution series of Xenopus egg extract was analyzed by SDS-PAGE and Western blotting. The indicated rabbit polyclonal antibodies were used to detect Nups. These primary antibodies were then decorated via either IRDye 800–labeled goat anti–rabbit polyclonal IgG (1:5,000; LI-COR Biosciences) or anti–rabbit IgG nanobody TP897 (10 nM). Blots were analyzed with an Odyssey Infrared Imaging System (LI-COR Biosciences). (b) Left: A twofold dilution series of HeLa cell lysate was analyzed by SDS-PAGE and Western blotting. The indicated mouse IgG1 mAbs were decorated via either IRDye 800–labeled goat anti–mouse polyclonal IgG (1:1,340, 5 nM; LI-COR Biosciences) or anti–mouse IgG1 Fc nanobody TP1107 (5 nM). Right: A twofold dilution series of Xenopus egg extract was blotted and probed with anti-Nup62 mouse IgG1 mAb A225. It was then detected via IRDye 800–labeled goat anti-mouse polyclonal IgG (5 nM), anti–mouse IgG1 Fc nanobody TP1107 (5 nM), anti–mouse IgG1 Fab nanobody TP886 (5 nM), anti–mouse κ chain nanobody TP1170 (2.5 nM), or a combination of TP1107 and TP886 or TP1107 and TP1170. Blue pixels indicate signal saturation. (c) A dilution series of filamentous bacteriophages was blotted and probed with an anti–minor coat protein pIII mouse IgG2a mAb. It was then decorated via either IRDye 800–labeled goat anti-mouse polyclonal IgG (2.5 nM) or anti–mouse κ chain nanobody TP1170 (2.5 nM). (d) Dual-color Western blotting. A twofold dilution series of Xenopus egg extract was blotted and probed with anti-Nup62 mouse IgG1 mAb A225 and rabbit anti-Nup54 polyclonal antibody. These primary antibodies were then detected via IRDye 800–labeled goat anti–rabbit polyclonal IgG and IRDye 680–labeled goat anti–mouse polyclonal IgG. Alternatively, they were detected with TP1107 coupled to IRDye 680 and TP897 coupled to IRDye 800.

Article Snippet: Polyclonal goat anti–mouse IgG coupled to IRDye 800CW (925-32210; LI-COR Biosciences) was used to detect primary mouse antibodies at a dilution of 1:1,340 (5 nM).

Techniques: Western Blot, Labeling, SDS Page, Imaging

Journal: PLoS ONE

Article Title: Aggregation of PolyQ Proteins Is Increased upon Yeast Aging and Affected by Sir2 and Hsf1: Novel Quantitative Biochemical and Microscopic Assays

doi: 10.1371/journal.pone.0044785

Figure Lengend Snippet: Proteins with polyQ of different lengths show different characteristics. A Schematic representation of the constructs used to express polyQ proteins. Each contains a FLAG tag at its N-terminus, followed by huntingtin's exon 1 encoding the first 17 amino acids (Htt), a polyQ tract of different length, and GFP at the C-terminus. Expression of all polyQ proteins is driven by the pGal1 promoter that is induced by galactose and repressed by glucose. B Wild-type cells (W303–1b) expressing the 25Q, 47Q or 103Q were grown logarithmically (0.6–1 A 600 ) for 8 hours under galactose induction. Cell lysates were resolved by SDS-PAGE and polyQ proteins were detected by immunoblotting (IB) with a mouse anti-FLAG antibody followed by IRDye 800CW-conjugated goat anti-mouse IgG (right panel) or a rabbit anti-GFP followed by Dylight 680-labeled goat anti-rabbit IgG (left panel, red). A mouse anti-actin followed by IRDye 800CW-conjugated goat anti-mouse IgG was used as a loading control (left panel, green). Blots were visualized by the Odyssey Infrared Imaging System. C Wild-type cells (W303–1b) expressing the 25Q, 47Q, 103Q or an empty plasmid were grown logarithmically in glucose and 10-fold serial dilutions (starting with 7.5×10 6 cells) were spotted on glucose or galactose plates.

Article Snippet: The secondary antibody used was IRDye 800CW-conjugated goat anti-mouse (LI-COR Biosciences).

Techniques: Construct, FLAG-tag, Expressing, SDS Page, Labeling, Imaging, Plasmid Preparation