Expressions of HIF-1α, total and phospho-T389 p70 S6K1, as well as total and phospho-S65 eIF4E-BP1 were determined by Western blot analysis and compared to β-actin in order to determine equal protein loading, as previously described [25 (link)]. Li-Cor goat secondary antibodies, conjugated with fluorescent dyes, were employed according to the manufacturer's protocol in order to visualise detected proteins (using a Li-Cor Odyssey imaging system). Following Western blot detection of phospho-T389 p70S6K1 and phospho-S65-eIF4E-BP1, membranes were stripped using a ReBlot™ Plus Kit (Chemicon International) according to the manufacturer's protocol. Membranes were then blocked and scanned using a Li-Cor Odyssey imaging system to make sure that stripping was successfully completed. Then membranes were re-probed with antibodies in order to detect the total amounts of p70S6K1 and eIF4E-BP1. Western blot data were quantitatively analysed using Odyssey software and values were normalised against respective β-actin bands.
Odyssey imaging system
Manufactured by LI COR
Sourced in United States, Germany, United Kingdom, China, Australia, Niger, Canada
The Odyssey Imaging System is a fluorescence-based imager designed for detection and quantification of proteins and nucleic acids. It utilizes two near-infrared fluorescent dyes to enable multiplex detection and analysis. The system can be used for a variety of applications, including Western blotting, gel and membrane-based assays, and microplate-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (114 mentions)
Pvdf membrane, by Merck Group (72 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (70 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (60 mentions)
Ripa buffer, by Thermo Fisher Scientific (59 mentions)
Protease inhibitor cocktail, by Roche (58 mentions)
Nitrocellulose membrane, by Bio-Rad (57 mentions)
Irdye 800cw, by LI COR (48 mentions)
Image studio software, by LI COR (39 mentions)
Protease inhibitor cocktail, by Merck Group (38 mentions)
β actin, by Merck Group (32 mentions)
Nitrocellulose membrane, by Merck Group (32 mentions)
Image studio lite software, by LI COR (31 mentions)
Pvdf membrane, by Bio-Rad (30 mentions)
Irdye 800, by LI COR (28 mentions)
Ripa buffer, by Merck Group (28 mentions)
Gapdh, by Merck Group (28 mentions)
Irdye 680rd, by LI COR (27 mentions)
β actin, by Cell Signaling Technology (26 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (26 mentions)
2 037 protocols using odyssey imaging system
1
Quantifying HIF-1α and mTOR Pathway Activation
2
Proteinase K Digestion of Alpha-Synuclein
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Samples with 4 μg of OW, OP, PFF, and pPFF were incubated at 37 °C in PK solution (1 μg/mL of PK [Sigma-Aldrich Corporation, St. Louis, MO, USA] in 20 mM Tris, pH 7.4, 0.15 NaCl) for 0, 5, 10, 20, and 40 min. The enzymatic reaction was terminated by the addition of sample buffer containing sodium dodecyl sulfate (SDS) and dithiothreitol and then boiled at 95 °C for 5 min. Afterward, the proteins were separated by a SDS-Tris 12% gel and transferred to a polyvinylidenedi fluoride (PVDF) membrane (Millipore). Finally, α-syn and p-α-syn was visualized in an Odyssey imaging system (Li-Cor, Lincoln, NE, USA) using an α-syn (ab138501; Abcam, Cambridge, MA, USA) and p-α-syn (pSyn#64; Wako Pure Chemical Industries, Ltd.) monoclonal antibody followed by a secondary antibody (926-68070, 926-68071; dilution, 1:10,000; LI-COR Biosciences, Lincoln, NE, USA). Finally, the membranes were analyzed using an Odyssey imaging system (Li-Cor).
3
Coomassie Gel Imaging and Western Blot
4
Western Blot Analysis of Viral Proteins
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Proteins from cell lysates were separated by 10% acrylamide SDS–PAGE and subjected to western blot analysis using polyclonal rabbit anti-NP antibody (catalogue number: PA5-32242; Thermo Fisher; diluted 1:2,500), monoclonal mouse anti-β-tubulin antibody (catalogue number: T4026; Sigma-Aldrich; diluted 1:1,000) and the corresponding secondary antibodies IRDye 680RD goat anti-rabbit IgG (H+L) (LI-COR Biosciences; diluted 1:5,000) or IRDye 800CW goat (polyclonal) anti-mouse (LI-COR Biosciences; diluted 1:5,000). Signals were visualized by Odyssey Imaging System (LI-COR Biosciences). Proteins of purified viral particles were separated as described above and detected by western blot analysis using polyclonal rabbit anti-NP, polyclonal mouse anti-M1 (provided by Dr. Jovan Pavlovic; diluted 1:100) and corresponding peroxidase-conjugated goat anti-mouse IgG (H+L) (Jackson ImmunoResearch; diluted 1:5,000) or peroxidase-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch; diluted 1:5,000) antibodies. Signals were detected with an Odyssey Imaging System (LI-COR Biosciences). Uncropped western blots can be found in Supplementary Fig. 9 .
5
Protein Extraction and Western Blot Analysis
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Mouse proteins were extracted from vaginal tissues using SDS extraction buffer (50 mM Tris pH 7.5, 0.5% SDS, Halt Protease inhibitor (Pierce, Rockford, IL) for 1.5 hours on ice after vortexing with 0.5 mm glass beads (Next Advance, Averill Park, NY) five times with 1 min vortexing and 1 min rests. Protein concentration was measured using a Nanodrop spectrophotometer (Thermo Scientific, Rockford, IL) and BCA assay (Pierce, Rockford, IL). Protein lysates (50 µg) were resolved on a 10% SDS-Page gel. The gel was transferred (wet transfer at 300 mA) to a nitrocellulose membrane and then blocked with 5% non-fat dry milk in 1× PBS. For the detection of CD166 antigen in mouse samples, the primary antibody was applied in blocking buffer overnight at 4°C: Secondary antibody was used incubated at room temperature for 1 hour. Following extensive washing, the membranes were scanned using the LI-COR Odyssey imaging system.
To detect OLFM-4 and β-actin, Vk2 cell extracts were resolved on 4–12% Bis-Tris SDS-PAGE gels and mucin 5B was resolved on 3–8% Tris-acetate SDS-PAGE gels f. Gels were transferred to PDVF membranes and probed with the appropriate primary and secondary antibodies and detected using the LI-COR Odyssey imaging system.
To detect OLFM-4 and β-actin, Vk2 cell extracts were resolved on 4–12% Bis-Tris SDS-PAGE gels and mucin 5B was resolved on 3–8% Tris-acetate SDS-PAGE gels f. Gels were transferred to PDVF membranes and probed with the appropriate primary and secondary antibodies and detected using the LI-COR Odyssey imaging system.
6
Western Blot Analysis of SEPT2 Protein
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Cells were lysed in buffer containing 50 mM Hepes, pH 7.4, 100 mM NaCl, 0.5% Triton X-100, 1 mM MgCl2, 1 mM PMSF, and protease inhibitors. Samples were centrifuged at 8,000 rpm for 10 min at 4°C, and the supernatants were collected. Equal volumes were resolved with a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane at 100V for 1 h at 4°C. Membranes were blocked with 5% dried milk (wt/vol) in PBS containing 0.1% Tween-20. A rabbit antibody against SEPT2 (1:5,000; Sigma-Aldrich) and a secondary anti-rabbit antibody (LI-COR Biosciences) conjugated to infrared dye was diluted in PBS containing 2% BSA, 0.1% Tween-20, and 0.025% sodium azide and sequentially incubated on the membrane before scanning with the Odyssey imaging system (LI-COR Biosciences).
InFig. 5 , equal volumes of canine kidney cytosol samples were resolved on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were blocked in 5% dried milk (wt/vol) in PBS containing 0.1% Tween-20. Rabbit antibody against SEPT2 (1:5,000; Sigma-Aldrich), mouse anti-actin (1:5,000; Sigma-Aldrich), and anti-rabbit or -mouse secondary antibodies (LI-COR Biosciences) conjugated to infrared dyes were diluted in PBS containing 2% BSA, 0.1% Tween-20, and 0.025% sodium azide and sequentially incubated on each membrane before scanning with the Odyssey imaging system (LI-COR Biosciences).
In
7
Western Blot Protein Detection
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Proteins that were denatured in Laemmli buffer were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked in 5% nonfat milk in TBS-T buffer and incubated with primary antibody overnight at 4°C. The membranes were then washed and incubated with secondary HRP-conjugated antibodies or LI-COR fluorescently labeled secondary antibodies, followed by ECL detection or analysis with the LI-COR Odyssey Imaging System (LI-COR Biosciences). For the detection of biotinylated proteins, Western blot membranes were incubated overnight with IRDye Streptavidin 800CW at 1:1000 dilution (LI-COR Biosciences) and analyzed with the LI-COR Odyssey Imaging System.
8
Protein Expression Profiling in Cell Lysates
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Cells were lysed with RIPA buffer (25 mM Tris HCl (pH 7.6), 1% sodium deoxycholate, 150 mM NaCl and 0.1% SDS) containing protease inhibitor cocktail (Roche, Basel, Switzerland). Total cell lysates with 60–80 µg protein were boiled for five minutes in SDS-PAGE reducing buffer, and loaded to SDS-PAGE gels. Separated proteins in the gel were transferred onto a nitrocellulose membrane. Primary antibodies used for immunoblotting were against androgen receptor (#3125, Cell Signaling Technology), vimentin (#5741, Cell Signaling Technology), fibronectin (15613-1-AP, Proteintech), Keratin 8/18 (#4546, Cell Signaling Technology), and GAPDH (#2118S, Cell Signaling Technology). Fluorescence-labeled secondary anti-mouse IgG antibody (LI-COR Biosciences, Lincoln, NE, USA) was used to detect the primary antibodies. The fluorescence intensity of the secondary antibody was assayed using an Odyssey Imaging System (LI-COR).
9
Immunoblot Analysis of ASCT2 Protein
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Immunoblot analysis was performed as previously described (22 (link)). Briefly, 2 million cells were pelleted and lysed in RIPA lysis and extraction buffer (ThermoFisher Scientific). Protein extracts were separated by electrophoresis on 12% SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (EMD Millipore). Membranes were blocked in 5% nonfat milk in PBS buffer and incubated with the rabbit anti-ASCT2 antibody [1:1,000, 8057S, Cell Signaling Technology (CST)]. Detection of GAPDH was used as a protein loading control. Immunoreactive bands were detected with horseradish peroxidase (HRP) anti-rabbit antibodies using the ECL system. Analysis of immunoblotting was performed using the LI-COR Biosciences Odyssey Imaging System.
10
Western Blot Protein Analysis
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Cells were lysed with cell lysis buffer at 4 °C for 30 min and centrifuged (12 000 rpm, 10 min, 4 °C). Protein concentrations of supernatants were measured with BCA assay (ThermoFisher Scientific). 30 μg of protein per sample was subjected to SDS-PAGE analysis and electro-transferred to methanol-activated immobilon-FL PVDF membranes (EMD Millipore, Billerica, MA). Membranes were blocked with 5% milk in TBST buffer and probed with primary antibodies with dilutions according to the manufacturer's instructions, subsequently with Dylight 800-conjugated secondary antibodies (ThermoFisher Scientific). The fluorescent signal was detected using the Odyssey Imaging System (LI-COR Biosciences).
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