Samples were denatured in SDS loading buffer and heated at 95 °C for 5 min. Proteins were resolved via SDS-PAGE and transferred to nitrocellulose membranes (BioRad, Hercules, CA). Membranes were blocked with Odyssey Blotting Buffer (Li-Cor Biosciences, Superior, NE) for 45 min at room temperature and then incubated with primary antibodies overnight at 4 °C as follows: H3 (Cell Signaling Technologies, Danvers, MA, 1:5000); H2B (Cell Signaling Technologies, 1:2000); H2A (Abcam, Cambridge, MA, 1:2000); H4 (Abcam, 1:1000); pan-acetyl lysine (Abcam 1:2500); pan-trimethyl lysine (PTM biolabs, Chicoago, IL, 1:5000); pan-butyryl lysine (PTM biolabs, 1:1000). Following three washes with Tris-buffered saline + 0.1% Tween-20 (TBST), infrared secondary antibodies (Li-Cor) were added in blocking buffer (1:5000) for 45 min. Blots were developed following 3 additional washes with TBST using the Odyssey Infrared Imaging System (Li-Cor).
Odyssey blotting buffer
Manufactured by LI COR
Odyssey Blotting Buffer is a buffer solution designed for use in Western blotting applications. It is formulated to provide the necessary ionic conditions for the transfer of proteins from a gel to a membrane during the blotting process.
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Lab products found in correlation
Infrared secondary antibody, by LI COR (3 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Azure c600 imaging system, by Azure Biosystems (2 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Pkm1 2, by Cell Signaling Technology (1 mentions)
Aldoa, by Cell Signaling Technology (1 mentions)
Cox 2, by Cell Signaling Technology (1 mentions)
H3k18la, by PTM Biolabs (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
3 protocols using odyssey blotting buffer
1
Western Blot Analysis of Histone Modifications
2
Glycolytic Enzyme Immunoblotting Assay
3
Western Blot Analysis of Protein Modifications
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Samples were denatured in SDS loading buffer and heated at 95 °C for 5 min. Proteins were then resolved via SDS-PAGE and transferred to nitrocellulose membranes (Biorad). Membranes were blocked with Odyssey Blotting Buffer (Li-Cor Biosciences, Lincoln, NE) for 45 min at room temperature. Primary antibodies were incubated with membranes overnight at 4 °C as described: GLO1 (1:1,000, Protein Technologies, 15140), ARG1 (1:1,000, Gentex, GTX109242), STAT3 (1:1,000, Cell Signaling Technologies, 4904S), GLO2 (1:1,000, ThermoFisher, PA5-28292), iNOS (1:2,000, Cell Signaling Technologies, #2024), COX-2 (1:2,000, Cell Signaling Technologies, #3582), H3K18La (1:1,000, PTMBio, PTM-1406), LactoylLys (1:500, PTMBio, PTM-1401), AcetylLys (1:500, Abcam, Ab21623), H3 (1:20,000, Cell Signaling Technologies, #3638S), Actin (1:10,000, Sigma, #A1978). Following 3x washes with TBS +0.1 % Tween-20 (TBST), infrared secondary antibodies (Li-COR) were added in blocking buffer (1:5,000) for 45 min. Blots were developed following 3 additional washes with TBST using a c600 Azure Imaging System (Azure Biosystems, Dublin, CA).
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