Plasma samples were diluted with PBS (1:2 dilution for mouse plasma, and 1:5 dilution for human plasma). 2 µl of the diluted plasma was separated under denaturing conditions on 4-15% SDS-PAGE (Bio-Rad) and transferred to PVDF membranes (Millipore). Membranes were incubated with MD-2 primary antibody (Abcam) and fluorescent secondary antibodies (LI-COR). Fluorescence was quantitated with an Odyssey Image System (LI-COR). An IgG western blot or a total protein stain using AzureRed Fluorescent Total Protein Stain (Azure Biosystems) was used as a loading control.
Odyssey image system
Manufactured by LI COR
Sourced in United States
The Odyssey Imaging System is a versatile and powerful instrument designed for high-sensitivity quantitative fluorescence detection and imaging. It provides accurate and reproducible results for a wide range of applications, including Western blotting, cell-based assays, and small animal imaging.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (3 mentions)
Protease inhibitor, by Roche (3 mentions)
Chemidoc image system, by Bio-Rad (3 mentions)
Irdye 800cw infrared dye, by LI COR (2 mentions)
Irdye 680rd infrared dye, by LI COR (2 mentions)
Sc 21733, by Santa Cruz Biotechnology (2 mentions)
Irdye 680cw goat anti mouse, by LI COR (2 mentions)
Bca assay, by Bio-Rad (2 mentions)
Irdye 800cw goat anti rabbit, by LI COR (2 mentions)
Protein assay reagent, by Bio-Rad (2 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Image studio lite software, by LI COR (2 mentions)
Fluorescent secondary antibody, by LI COR (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Azurered fluorescent total protein stain, by Azure Biosystems (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Irdye 800cw goat anti rabbit igg secondary antibody, by LI COR (1 mentions)
29 protocols using odyssey image system
1
Quantitative Analysis of MD-2 Protein in Plasma
2
Tissue and Cell Western Blot Procedure
3
Picornavirus Infection Cell Staining
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For cell staining, cells were plated at 1 × 104 cells/well in 96-well plates and incubated overnight. The cells were then infected with PeV-A3 at multiplicity of infection (MOI) = 5. After 8- and 24-h infection, the cells were fixed with 1:1 Methanol/Ethanol solution (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Cells were incubated with blocking buffer [10% skim milk in phosphate-buffered saline (PBS)] and incubated with anti-PeV VP0 polyclonal antibody at 4°C overnight (LTK BioLaboratories, Taoyuan, Taiwan) (10 (link), 29 (link)). Then, the IRDye® 800CW goat anti-rabbit IgG secondary antibody was added into the cells (Li-COR, #926-32211, Lincoln, NE, USA), followed by 2-h incubation. Images of immunofluorescence assay were captured, and the fluorescence intensity of PeV-A3-infected cells were quantified using Odyssey image system (Li-COR, Lincoln, NE, USA).
4
Protein Separation and Immunoblotting
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The purified protein or antibody was separated by electrophoresis in a 7.5%–12% polyacrylamide gel. The separated protein or antibody was revealed either using Coomassie blue or transferred to PVDF membrane for WB analysis under reducing or non-reducing conditions with β-mercaptoethanol. The membrane was first blocked and then incubated overnight at 4°C or 37°C for one hour with diluted plasma or antibody, followed by incubation with the secondary antibody of either anti-human IgG or anti-rabbit IgG conjugated with an IRDye 800CW (cat.# 926-32232, Rockland). Protein bands were visualized using the Odyssey Image System (Li-COR).
5
Western Blot Analysis of Protein Modifications
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Key resources table contains information of primary and secondary antibodies used in this study. For Western blot analysis, protein samples were separated by precast 4–15% Criterion™ TGX Stain-Free™ Protein Gels (Bio-Rad Laboratories 5678085), transferred to a nitrocellulose membrane using Trans-Blot® Turbo™ Midi Nitrocellulose Transfer Packs (Bio-Rad Laboratories 1704159EDU) and Trans-Blot® Turbo™ Transfer System (Bio-Rad Laboratories 1704150). The membranes were blocked with 5% non-fat dried skim milk solution for 1 hat 27°C, then incubated overnight at 4°C with primary antibodies diluted in blocking buffer: anti-OGT(1:10,000), anti-O-GlcNAc(1:10,000) and anti-GFP (1:10,000). The next day, fluorescent secondary antibodies (IRDye 800CW donkey anti-rabbit 800(1:20,000), IRDye 800CW donkey anti-mouse 680(1:20,000), IRDye 800CW goat anti-rabbit 800 (1:20,000) and IRDye 800CW goat anti-mouse 680 (1:20,000)), diluted in 1% milk in TBS-T, were added to the membranes for 1 hat room temperature. The membranes were washed 3x in TBS-T for 10 min each and subsequently images were acquired and analyzed using the LI-COR Odyssey Image System.
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6
Protein Extraction and Western Blot Analysis in Mice
7
Proteomic Analysis of Notch Signaling
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Cell lysates were separated by SDS PAGE and transferred to nitrocellulose membranes. Western blot analysis was performed by using primary antibodies against ASPH; JAG1, JAG2, DLL1, DLL4, Notch1, Notch2, Notch3, Notch4, c-Myc, matrix metalloproteinase (MMP)-2, epithelial cell adhesion molecule (EpCAM), and CD44 (Cell Signaling Technology #2620, #2205, #2588, #2589, #4380, #4530, #5276, #2423, #5605, #13132, #2929, #5640, respectively); activated Notch1, HES1 and HEY1 (Abcam ab8925, ab71559, ab22614, respectively); cyclin D3, MMP9, and proliferating cell nuclear antigen (PCNA) (Santa Cruz, sc-56307, sc-21733, sc-7907, respectively). Protein bands were visualized by IRDye® 680RD Infrared Dye and IRDye® 800CW Infrared Dye and exposed on Odyssey image system (LI-COR).
8
Western Blot Analysis of Neuroinflammatory Markers
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Frozen brain tissues or cultured cells were homogenized in cold RIPA buffer with protease inhibitors and proteins were quantified with BCA assay (BioRad, Mississauga, ON, Canada). Proteins were loaded at 50 μg per lane on a 12% polyacrylamide gel. Proteins were transferred to nitrocellulose membrane and then blocked with LiCor blocking buffer. Blots were further incubated with primary antibodies overnight at 4 °C on a shaker. Primary antibody used for Iba1 (1:500, rabbit, Wako), CD68 (1:500, mouse monoclonal, Dako), caspase-1 (1:1000, rabbit, Abcam), NLRP3 (1:1000, rabbit, Millipore), and β-actin (1:10,000 mouse, Sigma-Aldrich). IRDye 800CW goat anti-rabbit and IRDye 680CW goat anti-mouse (LiCor, 1:10,000) were used as secondary antibodies. Blots were imaged using LiCor Odyssey image system.
9
Western Blot Analysis of Protein Samples
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Frozen brain tissues or cultured cells were homogenized in cold RIPA buffer with protease inhibitors and proteins were quantified with BCA assay (BioRad, Mississauga, ON, Canada). Proteins were loaded at 50 μg per lane on a 12% polyacrylamide gel. Proteins were transferred to nitrocellulose membrane and then blocked with Odyssey blocking buffer. Blots were further incubated with primary antibodies overnight at 4 °C on a shaker. IRDye 800CW goat anti-rabbit and IRDye 680CW goat anti-mouse (Li-Cor, 1:10,000) were used as secondary antibodies. Blots were imaged using Odyssey image system (Li-Cor).
10
Western Blot Protein Analysis Protocol
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Total protein was extracted from the cells and prepared with RIPA buffer. Nuclear or cytoplasmic proteins were isolated as described above. Protein samples were boiled for 5 min at 95 °C. The protein concentrations were quantified by the BCA protein assay. In brief, samples with the same amount of proteins were separated on 10% polyacrylamide gel. The proteins were then electrophoretically transferred at 15 V at RT onto a NC membrane. The blotted membranes were blocked in 5% nonfat dry milk in TBST for 1 h and then incubated overnight at 4 °C with the indicated primary antibodies. After three washes with TBST, the membranes were then incubated with corresponding secondary antibodies for 1 h and bands were visualized and analyzed by the LI-COR odyssey image system (Lincoln, NV, USA). Each Western blot analysis was performed in triplicate [42 (link)].
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