Western blot analysis was performed as described previously with minor modification.16 (link),28 (link) For cPLA2 expression, primary antibodies included mouse monoclonal anti-cPLA2 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal rabbit anti–phospho(p)-cPLA2 antibody (1:500), and mouse anti–β-tubulin antibody (1:1,000; Sigma). For active caspase-3 and poly(adenosine diphosphate ribose) polymerase (PARP) expression, primary antibodies included rabbit anti–caspase-3 antibody (cleaved, 1:1,000), rabbit anti–PARP-1 (cleaved, 1:500), and mouse anti–β-tubulin antibody (1:1,000; Sigma). For extracellular signal-regulated kinase (ERK) expression, primary antibodies included polyclonal rabbit anti-ERK1/2 antibody (1:1,000) and monoclonal mouse anti–p-ERK1/2 antibody (1:2,000). Secondary Alexa Fluor 680 goat antimouse (1:10,000; Invitrogen, Grand Island, NY) and IRDye 800 goat antirabbit (1:5,000; Rockland, Gilbertsville, PA) antibodies were used. The Western blot was imaged and quantified using a Li-Cor Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln, NE) according to the manufacturer's instruction.
Irdye 800 goat antirabbit
Manufactured by Rockland Immunochemicals
The IRDye 800 goat anti-rabbit is a near-infrared dye-conjugated secondary antibody. It is designed for use in western blotting, ELISA, and other immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (3 mentions)
Alexa fluor 680 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Mouse anti β tubulin antibody, by Merck Group (1 mentions)
Goat anti rabbit irdye 680, by LI COR (1 mentions)
Proteinase inhibitor tablet, by Roche (1 mentions)
β actin, by Merck Group (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor mixture, by Thermo Fisher Scientific (1 mentions)
3 protocols using irdye 800 goat antirabbit
1
Quantitative Western Blot Analysis
2
Histone Extraction and Western Blot
3
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The cells were lysed with RIPA buffer containing 0.1% SDS, 1% Triton-X 100, 10% glycerol, 150 mM NaCl, 0.05% Na-Doc, 5 mM EDTA (pH 8.0), 30 mM Tris-HCl (pH 7.4), protease inhibitor mixture (Thermo Fisher Scientific) and phosphatase inhibitors (Roche). After centrifugation (12,000 rpm, 4°C, 15 min) to remove cell debris, the protein suspension was collected and separated by SDS-PAGE. Then, the target proteins were transferred onto PVDF membranes (Millipore Durapore, 0.45 μm pore size). The membranes were washed, blocked and incubated with the specific primary antibodies (1:1000). The secondary antibody was IRDye 800 goat anti-rabbit or IRDye 700 goat anti-mouse (Rockland). Finally, the fluorescence signal of blots was detected using the Odyssey Infrared Imaging System (LI-COR, Lincoln, USA).
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