Cells were lysed in lysis buffer (20 mM Tris-HCl, pH 6.8, 137 mM NaCl, 1 mM EGTA, 1% Triton x100, 10% Glycerol and protease inhibitors cocktail) and protein samples were boiled in Laemmli buffer for 5–7 min at 100°C, separated by SDS-PAGE, transferred onto PVDF membranes, subjected to western blot analysis, and visualized using an ECL enhanced chemiluminescence detection kit (GE Healthcare), or with direct infrared fluorescence detection on an Odyssey Infrared Imaging System.
Odyssey infrared imaging system
Manufactured by GE Healthcare
The Odyssey Infrared Imaging System is a versatile laboratory instrument designed for high-performance protein and nucleic acid detection and quantification. The system utilizes infrared fluorescence technology to enable sensitive and accurate detection of a wide range of biomolecules.
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Lab products found in correlation
6 protocols using odyssey infrared imaging system
1
Protein Extraction and Western Blotting
2
Protein Extraction and Western Blotting
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Cells were lysed in lysis buffer (20 mM Tris-HCl, pH 6.8, 137 mM NaCl, 1 mM EGTA, 1% Triton x100, 10% Glycerol and protease inhibitors cocktail) and protein samples were boiled in Laemmli buffer for 5–7 min at 100°C, separated by SDS-PAGE, transferred onto PVDF membranes, subjected to western blot analysis, and visualized using an ECL enhanced chemiluminescence detection kit (GE Healthcare), or with direct infrared fluorescence detection on an Odyssey Infrared Imaging System.
3
Western Blot Protein Extraction and Analysis
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Cells were washed with ice-cold PBS and directly lysed with 2× Laemmli buffer and boiled at 100 °C for 10 min or lysed with RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate monohydrate, 0.1% SDS, supplemented with protease and phosphatase inhibitors cocktails (Roche)). When lysed in RIPA buffer, cells were incubated on ice for 10 min, centrifuged at 16,100 g for 10 min and protein concentration of supernatants was determined using a Bradford assay kit (Bio-Rad). Lysates were then denatured with 2× Laemmli buffer and boiled at 100 °C for 10 min, separated by SDS-PAGE, transferred onto PVDF membranes, subjected to western blot analysis, finally visualized using an ECL enhanced chemiluminescence detection kit (GE Healthcare), or with direct infrared fluorescence detection on an Odyssey Infrared Imaging System. Densitometric analysis on the immunoblots was performed using ImageJ program or IMAGE STUDIO Lite software.
4
Mitochondrial tRNA Detection by Northern Blot
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RNA (5 μg) was resolved on 1.2% agarose formaldehyde gels, then transferred to 0.45 μm Hybond-N+ nitrocellulose membrane (GE Lifesciences) and hybridized with biotinylated oligonucleotide probes specific to mitochondrial tRNAs. The hybridizations were carried out overnight at 50°C in 5× SSC, 20 mM Na2HPO4, 7% SDS and 100 μg ml−1 heparin, followed by washing. The signal was detected using either streptavidin-linked horseradish peroxidase or streptavidin-linked infrared antibody (diluted 1:2000 in 3× SSC, 5% SDS, 25 mM Na2HPO4, pH 7.5) by enhanced chemiluminescence (GE Lifesciences) or using an Odyssey Infrared Imaging System.
5
Western Blot Analysis Protocol
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Cells were lysed in Laemmli sample buffer and boiled for 10 min at 100°C, separated by SDS-PAGE, transferred to PVDF membranes and developed with primary and secondary antibodies. Primary antibodies were used with overnight incubation at 4°C, unless otherwise stated, and the secondary antibodies are used at a concentration of 1:2000 and incubated for 1 h at room temperature. For immunoprecipitation experiments, light-chain specific secondary antibodies were used at a 1:1000 dilution for 1 h at room temperature. Blots were developed using an ECL enhanced chemiluminescence detection kit (GE Healthcare), or with direct infrared fluorescence detection on an Odyssey Infrared Imaging System. Western blots in main Figures 1a-c. 1h, 3a, 3c-e, 4a-c, and figures in extended data 1e, 3c, 5a, 5d, 5g, 5a and 6f were developed with chemiluminescence, while the other blots were developed with fluorescence (LICOR Odyssey system). Densitometric analysis on the immunoblots was performed using IMAGE STUDIO Lite software, which enables quantitative analysis of blotting signals.
6
Mitochondrial Transcript Detection via Northern Blot
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RNA (5 µg) was resolved on 1.2% agarose formaldehyde gels, then transferred to 0.45 µm Hybond-N + nitrocellulose membrane (GE Lifesciences) and hybridized with biotinylated oligonucleotide probes specific to mitochondrial mRNAs, rRNAs and tRNAs. The hybridizations were carried out overnight at 50°C in 5x SSC, 20 mM Na 2 HPO 4 , 7% SDS and 100 µg.ml -1 heparin, followed by washing. The signal was detected using either streptavidinlinked horseradish peroxidase or streptavidin-linked infrared antibody (diluted 1: 2000 in 3x SSC, 5% SDS, 25 mM Na 2 HPO 4 , pH 7.5) by enhanced chemiluminescence (GE Lifesciences) or using an Odyssey Infrared Imaging System.
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