AGS cells were transfected with the hsa-miR-124-3p or negative control miRNA mimic for 24 or 48 h, collected, and quick-frozen for analysis. Spermine oxidase activity was measured using a luminol-based assay measuring the production of hydrogen peroxide, as previously described.44 (link) Polyamine concentrations were determined as previously described.45 (link) Both assays were normalized relative to milligrams of total cellular protein determined using the method of Bradford.46 (link)For Western blots, total protein (50 μg per lane) was separated on pre-cast 4–12% Bis-Tris NuPAGE gels with 1 × MOPS running buffer (Invitrogen) and transferred onto Immun-Blot PVDF membranes (BioRad). Blots were blocked for 1 hour at room temperature in Odyssey blocking buffer (LI-COR, Lincoln, NE, USA), followed by overnight incubation at 4°C with antibodies specific to SMOX (1:1000 dilution), as previously described,2 (link) and β-actin (#sc-8432, Santa Cruz Biotechnology, Dallas, TX, USA). Incubation with species-specific, fluorophore-conjugated secondary antibodies allowed the visualization and quantification of immunoreactive proteins using the Odyssey infrared detection system and software (LI-COR).
Odyssey infrared detection system and software
Manufactured by LI COR
The Odyssey infrared detection system and software is a product offered by LI-COR. It is designed for quantitative near-infrared fluorescence detection and analysis. The system includes hardware, software, and related accessories to enable researchers to perform various imaging and analytical tasks.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (4 mentions)
Immun blot pvdf membrane, by Bio-Rad (3 mentions)
Mops running buffer, by Thermo Fisher Scientific (3 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Dc assay, by Bio-Rad (2 mentions)
Immun blot pvdf, by Bio-Rad (1 mentions)
γ h2ax, by Abcam (1 mentions)
Bolt bis tris gel, by Thermo Fisher Scientific (1 mentions)
P s6kt389, by Cell Signaling Technology (1 mentions)
Raptor, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
5 protocols using odyssey infrared detection system and software
1
Evaluating miR-124-3p Effects on Spermine Oxidase
2
Quantitative Western Blot Analysis of Polyamine Metabolic Enzymes
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Total protein was extracted from treated cells and quantified using the Bio-Rad DC assay with absorbance measured at 750 nm; values were converted to protein concentration using interpolation on a bovine serum albumin standard curve. Protein samples (30 μg per lane) were separated on pre-cast 4–12% Bis-Tris BOLT gels with 1 × MOPS running buffer (Invitrogen) and transferred onto Immun-Blot PVDF membrane (Bio-Rad). Blots were blocked for 1 hour at room temperature in Odyssey blocking buffer (LI-COR, Lincoln, NE), and proteins of interest were visualized using antibodies specific to SSAT [46 (link)], SMOX [45 (link)], and ß-actin (Santa Cruz Biotechnology, Dallas, TX). Primary antibodies were diluted 1:1000 in Odyssey blocking buffer containing 0.1% Tween-20, applied to blocked membranes, and incubated at 4°C overnight with rocking. Following washes, blots were incubated for 1 hour at room temperature with species-specific, fluorophore-conjugated secondary antibodies to allow visualization and quantification of immunoreactive proteins using the Odyssey infrared detection system and software (LI-COR).
3
Evaluating miR-124-3p Effects on Spermine Oxidase
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AGS cells were transfected with the hsa-miR-124-3p or negative control miRNA mimic for 24 or 48 h, collected, and quick-frozen for analysis. Spermine oxidase activity was measured using a luminol-based assay measuring the production of hydrogen peroxide, as previously described.44 (link) Polyamine concentrations were determined as previously described.45 (link) Both assays were normalized relative to milligrams of total cellular protein determined using the method of Bradford.46 (link)For Western blots, total protein (50 μg per lane) was separated on pre-cast 4–12% Bis-Tris NuPAGE gels with 1 × MOPS running buffer (Invitrogen) and transferred onto Immun-Blot PVDF membranes (BioRad). Blots were blocked for 1 hour at room temperature in Odyssey blocking buffer (LI-COR, Lincoln, NE, USA), followed by overnight incubation at 4°C with antibodies specific to SMOX (1:1000 dilution), as previously described,2 (link) and β-actin (#sc-8432, Santa Cruz Biotechnology, Dallas, TX, USA). Incubation with species-specific, fluorophore-conjugated secondary antibodies allowed the visualization and quantification of immunoreactive proteins using the Odyssey infrared detection system and software (LI-COR).
4
Protein Quantification and Western Blot Analysis
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Following treatment, cells were lysed in 4% SDS containing protease inhibitors and passed through a homogenizer column (Zymo Research, Irvine, CA). Protein was quantified using the BioRad DC assay with interpolation on a bovine serum albumin standard curve. Reduced samples (30 μg/lane) were separated on 4–12% Bis-Tris BOLT gels (Invitrogen), followed by transfer onto Immun-Blot PVDF (BioRad) and blocking in Odyssey blocking buffer (LI-COR, Lincoln, NE) at room temperature for 1 hour. Membranes were incubated with primary antibodies targeting SMOX, SSAT, ODC, γH2AX (Abcam, Cambridge, MA), and ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Species-specific, fluorophore-conjugated secondary antibodies were used for visualization and quantitation of bands using an Odyssey infrared detection system and software (LI-COR).
5
Western Blot Analysis of Protein Markers
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