Lysates were prepared from liver tissue and protein levels were determined by the bicinchoninic acid assay (Thermo Fisher, Carlsbad, CA). A total of 50 μg protein was applied to 10% SDS-PAGE gel, then transferred to PVDF membranes. The membranes were probed overnight at 4 °C with antibodies directed against PGC1α (#101707, Cayman Chemical, Ann Arbor, MI, 1:500) or GAPDH (MAB374, Millipore, Temecula, CA, 1:2000). After washing, membranes were probed with fluorescently-labeled secondary antibodies (Li-Cor, Lincoln, NE), and immunoreactive proteins detected using an Odyssey fluorescent scanner (Li-Cor, Lincoln, NE).
Odyssey fluorescent scanner
Manufactured by LI COR
Sourced in United States
The Odyssey Fluorescent Scanner is a high-performance imaging system designed for detection and quantification of fluorescent signals in a variety of applications, including Western blotting, cell-based assays, and tissue imaging. The scanner utilizes a laser-based detection method to capture images with high sensitivity and resolution, allowing for accurate and reliable analysis of fluorescent samples.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Irdye 800, by LI COR (2 mentions)
Iblot dry transfer system, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Fluorescently labeled secondary antibody, by LI COR (1 mentions)
Mab374, by Merck Group (1 mentions)
Dylight800, by Cell Signaling Technology (1 mentions)
Grip1, by BD (1 mentions)
Tris glycine gel, by Lonza (1 mentions)
Irdye 680, by LI COR (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Merck Group (1 mentions)
Tip sonicator, by Emerson (1 mentions)
Bis tris sds page gel, by Thermo Fisher Scientific (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
Anti nrf2, by Proteintech (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
9 protocols using odyssey fluorescent scanner
1
Liver Protein Quantification and Western Blot
2
Quantifying PARP10 de-ADP-ribosylation Kinetics
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Following a previously established protocol (23 ), auto-mono-ADP-ribosylated PARP10cd was obtained by incubating 10 μM purified PARP10cd in 1 mM NAD+ for 20 min at 37˚C in Reaction Buffer (50 mM HEPES pH 8.0, 0.15 M NaCl, 0.2 mM TCEP, 0.02% NP-40). The de-ADP-ribosylation reaction was performed by incubating purified RuV macrodomain with mono-ADP-ribosylated PARP10cd at 37˚C in Reaction Buffer at equimolar ratios (1 μM). The reaction was terminated at 0, 1, 2, 4, 8, 16, 32 or 64 min by adding SDS-PAGE sample loading buffer and incubating at 95˚ for 5 min. Following separation SDS-PAGE on a 4-20% gradient gel, the proteins were transferred to a PVDF membrane and the de-MARylation activity determined by Western blotting. The membrane was blocked in 5% milk in PBS, 0.05% Tween-20. The membrane was incubated for 3 h at 22˚C in Anti-mono-ADP-Ribose Binding Reagent (Sigma, MABE1076, RRID:AB_2665469; 1:3,000 dilution) in place of a primary antibody. The secondary antibody was anti-rabbit immunoglobin (Cell Signaling Technology, 5151S, RRID:AB_10697505, DyLight® 800; dilution 1:10,000 dilution, 30 min at 22˚C). Blots were imaged with the near-infrared system of an Odyssey fluorescent scanner (LI-COR Biosciences) after washing with PBS and water.
3
Quantifying GRIP1 in Prefrontal Cortex
4
Western Blot Analysis of Hippocampal CRFR2
5
Protein Extraction and Western Blot Analysis
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Cells were collected by scraping in 250 μL ice cold 1x RIPA buffer with protease inhibitor. Lysates were sonicated using a tip sonicator (Branson) followed by clearing of insoluble material by centrifugation. The protein concentration of the supernatants was estimated by absorbance measurements on a Nanodrop instrument. 30 μg of lysate per lane was resolved using 4–12% Bis-Tris SDS PAGE gels (Invitrogen) and then transferred to PVDF membrane (Bio-Rad). Membranes were blocked in 5% non-fat dry milk (Biorad) in TBST (Tris-buffered saline with 0.1% Tween 20) for 1 hr at room temperature. Primary antibodies were incubated in milk in TBST overnight at 4 °C. Primary antibodies used here were anti-TUBG1 (Sigma, T6557, 1:2000), FLAG (Sigma, M2, 1:2000), anti-NRF2 (Proteintech, 66504, 1:2000), anti-succinyllysine (PTM Biolabs, PTM-401, 1:1000), anti-MYC (and anti-SUCLG2 (Bethyl Laboratories, A305-533A, 1:2000). After 5x TBST washes for 15–30 minutes, fluorophore-conjugated (1:2000, LI-COR) or HRP-conjugated (1:3000, ThermoFisher) secondary antibodies were incubated in milk for 1 hr and then washed again with TBST before visualizing on a LI-COR Odyssey fluorescent scanner or with film.
6
Subcellular Protein Fractionation and Western Blot
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Cells were harvested with 2 mM EDTA in HBSS (Gibco) and washed twice with HBSS. Cell pellets were then lysed with Cell Lysis Buffer (CLB) (10 mM Hepes 7.6, 10 mM KCl, 1.5 mM MgCl2, 0.5% NP40, PMSF, protease/phosphatase inhibitors (HALT–Pierce). The cytosolic fraction was separated from the nuclear pellet. The nuclear pellet was lysed with Nuclear Lysis Buffer (NLB) (25 mM Tris 7.5, 1% SDS, protease/phosphatase inhibitors). Protein concentrations were determined using Pierce BCA Protein Assay.
Equivalent amounts of cytosolic or nuclear protein were loaded into wells of SDS–PAGE gels (4–12% Bis-Tris Gel, Thermo Fisher), and transferred to nitrocellulose. These membranes were incubated with the following antibodies at 4 °C overnight: Primary antibodies (Cell Signaling: ERK, phosphor-ERK, FGFR1-D8E4. Sigma: Actin). Secondary Antibodies (ThermoFisher: Goat anti Mouse Alexa Fluor 680, Goat anti Rabbit Alexa Fluor 800). Stained membranes were scanned with a LI-COR Odyssey Fluorescent Scanner.
Equivalent amounts of cytosolic or nuclear protein were loaded into wells of SDS–PAGE gels (4–12% Bis-Tris Gel, Thermo Fisher), and transferred to nitrocellulose. These membranes were incubated with the following antibodies at 4 °C overnight: Primary antibodies (Cell Signaling: ERK, phosphor-ERK, FGFR1-D8E4. Sigma: Actin). Secondary Antibodies (ThermoFisher: Goat anti Mouse Alexa Fluor 680, Goat anti Rabbit Alexa Fluor 800). Stained membranes were scanned with a LI-COR Odyssey Fluorescent Scanner.
7
Quantification of FoxO1 and Akt Signaling
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WB analysis was used to quantify the expression of FoxO1, p-FoxO1, Akt, p-Akt, GPR17, and GAPDH active in signaling. After different treatments, total protein from SK-N-SH cells was extracted at 4 °C and the protein concentration quantified using a BCA protein assay kit. For each sample, 40 μg of protein was loaded. The following primary antibodies were used: anti-FoxO1 antibody (1:1000, Cell Signaling #2880), anti-p-FoxO1 Ser256 antibody (1:1000, Cell Signaling #9461), anti-Akt antibody (1:1000, Cell Signaling #4691), anti-p-Akt Ser473 antibody (1:1000, Cell Signaling #9271), anti-GPR17 antibody (1:500, Absin Bioscience Inc. #abs112096, Shanghai, China), and anti-GAPDH antibody (1:5000, Abcam #181602). The second antibody used was Goat anti-Rabbit IgG H&L (IRDye® 800 CW; 1:10,000, Abcam #216773). An Odyssey fluorescent scanner (LI-COR, Bioscience, Lincoln, NE, USA) was used to detect protein bands. The relative fold-change of protein expression levels was calculated by normalization to GAPDH levels using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
8
Hippocampal Protein Analysis via Western Blot
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Mice were taken directly from their home cages and killed by cervical dislocation. Whole hippocampi were dissected and frozen in liquid nitrogen. Tissue was homogenized in 200 μL of ice-cold extraction buffer containing PBS, 1 mM EGTA, 1 mM EDTA, 0.01% SDS, and 1 mM PMSF. Protein concentration was quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein (30 μg for whole cell) were loaded and separated in 10% polyacrylamide Tris–Glycine gels (Invitrogen) and transferred to nitrocellulose membranes using the i-Blot dry transfer system (Invitrogen). Membranes were blocked with Li-Cor blocking buffer. Membranes were incubated for 24 h at 4°C with selective antibodies to: CREB (1:1000; Santa Cruz), pCREB (1:1000, Millipore), TORC1 (1:1000, Cell Signaling), and GAPDH (1:2000, Cell Signaling). Membranes were then incubated with fluorescent secondary antibodies (1:5000, IR-dye 680 or IR-dye 800), before being imaged on an Odyssey fluorescent scanner (Licor Biosciences). GAPDH was used as an internal loading control.
9
Histone H3K9 methylation in B cells
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B cells from four individuals with low KDM4C expression (GM11839, GM06994, GM12891, GM07034) and four individuals with high KDM4C expression (GM07056, GM12144, GM12815, GM12003) were washed in PBS and suspended in Triton lysis buffer (PBS with 0.5% Triton X 100 [v/v], 1× complete protease inhibitors [Roche], and 5 mM sodium butyrate) at 1 × 107 cells/mL and lysed for 10 min on ice. Cell nuclei were collected by centrifugation and resuspended in 250 μL of 0.2 M HCl and incubated overnight at 4°C to extract histones. The supernatant was collected, and 10–20 μg of protein were analyzed by Western blot with the following antibodies: 2 μg trimethylated H3K9 (17-625; Millipore), 2 μg dimethylated H3K9 (39683; Active Motif), 5 μg monomethylated H3K9 (39681; Active Motif), or 0.5 μg total H3 (Abcam). Quantitative Western blot detection was carried out using secondary antibodies coupled to fluorophores (Licor), and the fluorescent signal was detected on an Odyssey fluorescent scanner (Licor). A representative Western blot is shown in greyscale in Supplemental Figure S4. No significant difference in fluorescent signal was detected among individuals with high and low KDM4C expression for any of the methylation states of H3K9, by t-test. Histone extractions from B cells were repeated, and the same results were obtained.
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