After 48 h of in vitro stimulation, cells transfected with pCMV‐HBx were lysed. Proteins were separated using 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto polyvinylidene difluoride membranes. The membranes were incubated at 4°C with rabbit anti‐human IL‐35 (PeproTech) or EBI3 (ab83896, Abcam) overnight. Thereafter, the membranes were incubated with the IRDye 800 goat anti‐rabbit secondary antibody (LI‐COR Biosciences). Similarly, phosphor‐JNK and phosphor‐c‐Jun (all purchased from Cell Signaling Technology, Inc) were used to detect the phosphorylation of the JNK/c‐Jun pathway components. LI‐COR Odyssey was used to detect the target protein.
Irdye 800 goat anti rabbit secondary antibody
Manufactured by LI COR
Sourced in United States
IRDye 800 goat anti-rabbit secondary antibodies are infrared-labeled secondary antibodies designed for use in Western blotting, ELISA, and other immunodetection applications. These antibodies specifically bind to rabbit primary antibodies, enabling detection and visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (2 mentions)
Goat anti mouse irdye 680, by LI COR (2 mentions)
Phosphor jnk, by Cell Signaling Technology (1 mentions)
Phosphor c jun, by Cell Signaling Technology (1 mentions)
Imagequant 5, by GE Healthcare (1 mentions)
Enhanced chemi luminescence (ecl), by Advansta (1 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Sab4301138, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
As05084, by Agrisera (1 mentions)
5 protocols using irdye 800 goat anti rabbit secondary antibody
1
Investigating HBx-Mediated Cytokine Signaling
2
Western Blot Analysis of IGFBP2
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Plasma samples (2.5 µL of a 1:6 dilution) were mixed with equal volumes of 2× Laemmli buffer containing dithiothreitol and mercaptoethanol and boiled for 10 min.
They were electrophoresed on 13% SDS-PAGE gels and electroblotted onto nitrocellulose membranes (Protran, Schleicher & Schuell Bioscience, Keene, NH, USA). The membranes were blocked in Tris-buffered saline with Tween-20 (TBST; 0.05 M Tris, pH 7.4, 0.2 M NaCl, 0.1% Tween-20) containing 3% w/v nonfat dried skim milk (NFM) for 1 h at room temperature and incubated with 1:1000 dilution of bovine insulin-like growth factorbinding protein 2 (IGFBP2) antiserum (#06-107, Millipore) in the blocking buffer for 2 h at room temperature. After washes in TBST, membranes were incubated with 1:20,000 IRDye 800 goat anti-rabbit secondary antibody (LI-COR Biotechnology, Lincoln, NE, USA) in TBST with 3% NFM for 1 h at room temperature. Signals were quantified with the LI-COR Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA) using the 800-nm channel.
They were electrophoresed on 13% SDS-PAGE gels and electroblotted onto nitrocellulose membranes (Protran, Schleicher & Schuell Bioscience, Keene, NH, USA). The membranes were blocked in Tris-buffered saline with Tween-20 (TBST; 0.05 M Tris, pH 7.4, 0.2 M NaCl, 0.1% Tween-20) containing 3% w/v nonfat dried skim milk (NFM) for 1 h at room temperature and incubated with 1:1000 dilution of bovine insulin-like growth factorbinding protein 2 (IGFBP2) antiserum (#06-107, Millipore) in the blocking buffer for 2 h at room temperature. After washes in TBST, membranes were incubated with 1:20,000 IRDye 800 goat anti-rabbit secondary antibody (LI-COR Biotechnology, Lincoln, NE, USA) in TBST with 3% NFM for 1 h at room temperature. Signals were quantified with the LI-COR Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA) using the 800-nm channel.
3
Western Blot Analysis of Protein Expression
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Total cell lysates or nuclear and cytosolic fractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to PVDF-FL membranes. Blots were incubated with primary antibody overnight at 4°C, washed 3 times with 1X PBST then incubated in IRDye 680 goat anti-mouse or IRDye 800 goat anti-rabbit secondary antibodies (LI-COR, Lincoln, NE) for 1 hour, washed 3 times in 1X PBST and scanned with an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE) [51 (link)]. The expression levels of β-actin were used as the loading control for the Western blots. Immunoblot analyses were performed at least twice. Representative immunoblots were subjected to densitometry analysis. Densitometry was performed using ImageQuant 5.2 (GE Healthcare, Piscataway, NJ).
4
Western Blot Protein Profiling
5
Analyzing Arabidopsis Protein Expression
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One hundred and twenty mg of tissue of Arabidopsis transgenic lines overexpressing CV-FLAG were frozen in liquid N2 and grounded using a bead beater homogenizer. Proteins were extracted in an extraction buffer (100 mM Tris-Cl pH 7.5, 100 mM NaCl, 1 mM EDTA, 2% SDS, 1% protease inhibitor cocktail Sigma Cat. no. P9599) in a 4:1 w/v ratio. Samples were centrifuged at 16000 x g for 10 min at 4°C. Supernatants were transferred to fresh eppendorf tubes and stored at -80°C until use. Thirty µg of total proteins were separated by SDS-PAGE, transferred to a Hybond-N+ nylon membrane (GERPN203B), and probed as described by Wang and Blumwald (2014) (link). Antibodies raised against PsbO (AS05092), PsaB (AS06166A), PsbA/D1 (AS05084), GS1/GS2 and Lhcb2 (AS01003) were from Agrisera (Vännäs, Sweden). Antibodies raised against FLAG were from Sigma Aldrich (SAB4301138). IRdye 800 goat anti-rabbit secondary antibodies were obtained from LI-COR (926-32211). Horseradish peroxidase-conjugated goat anti-mouse secondary antibodies were purchased from Abcam (ab789).
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