Immunoblotting analysis was conducted as previously described using an Odyssey Infrared Image System (LI‐COR Biosciences (Zou et al., 2017 )). Antibodies against β‐tubulin (no. 2,146) were purchased from Cell Signaling and were diluted 1:1,000. UCP1 polyclonal antibody (no. PA1‐24894) and PR domain containing 16 polyclonal antibody (PRDM16; no. PA5‐20872) were purchased from Thermo Scientific and were diluted 1:1,000. Band density was quantified and then normalized to β‐tubulin content, because the levels of β‐tubulin did not differ between experimental groups.
Odyssey infrared image system
Manufactured by LI COR
Sourced in United States
The Odyssey infrared image system is a versatile and powerful tool designed for quantitative infrared fluorescence detection and analysis. This system utilizes advanced infrared imaging technology to capture high-resolution images of a wide range of sample types, including protein gels, Western blots, and cell-based assays. The Odyssey system is capable of detecting and quantifying multiple targets simultaneously, making it a valuable tool for a variety of research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (7 mentions)
Irdye 800cw goat anti rabbit, by LI COR (5 mentions)
Image studio lite software, by LI COR (4 mentions)
Dc protein assay kit, by Bio-Rad (4 mentions)
Immobilon fl membrane, by Merck Group (3 mentions)
Qproteome mammalian protein prep kit, by Qiagen (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Laemmli sample buffer, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Protease inhibitor tablet, by Thermo Fisher Scientific (3 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (3 mentions)
Phosphatase inhibitor tablet, by Thermo Fisher Scientific (3 mentions)
Infrared dye labeled respective secondary abs, by LI COR (3 mentions)
Anti tgf β, by Proteintech (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
β actin, by Merck Group (3 mentions)
Nupage mes sds running buffer, by Thermo Fisher Scientific (3 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Irdye 680 goat anti mouse secondary antibody, by LI COR (2 mentions)
Anti ip6k1, by GeneTex (2 mentions)
63 protocols using odyssey infrared image system
1
Immunoblotting Analysis of Thermogenic Proteins
2
Adipose Tissue Protein Analysis
3
Immunoblot Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoblot analysis, the fractional homogenate samples were prepared using reducing sample buffer (100 mM Tris-Cl (pH 6.8), 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol and 200 mM 1-4, dithiothreitol, DTT). Proteins in homogenates were separated using polyacrylamide gel electrophoresis and were identified using primary antibodies after transfer of proteins to nitrocellulose membranes using standard methods. The immunoblots were imaged using the Odyssey infrared image system (LICOR Inc.) and relative levels of immunoreactivity were analyzed using Image Studio TM Lite software (LICOR Inc.).
4
Multiplex Immunofluorescence Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sections were blocked in StartingBlock T20 (TBS) Blocking Buffer (Thermo Scientific, Waltham, MA) for 1 hour and subsequently transferred to the primary antisera (TH: Millipore Ab318, mouse anti-TH, 1:4,000) to incubate overnight at 4 °C. Following primary incubation, tissue was incubated in the dark in secondary antisera against mouse IgG (LI-COR 926–32212, donkey anti-mouse IRDye 800CW, 1:500) for 1 hour at room temperature. Tissue was then blocked in StartingBlock T20 (TBS) Blocking Buffer (Thermo Scientific) for 1 hour at room temperature before being incubated in the primary antisera against GFP (Abcam Ab290, rabbit anti-GFP, 1:100,000) overnight at 4 °C in the dark. Following primary incubation, tissue was incubated in secondary antisera against rabbit IgG (LI-COR 925–68021, goat anti-rabbit IRDye 680LT, 1:500) for 1 hour at room temperature. Sections were mounted on subbed slides, dehydrated via ascending ethanol washes, cleared with xylene, and coverslipped with Cytoseal. Multiplexed signal intensities were imaged with both 700 and 800 nm channels in a single scan with a resolution of 42 μm and depth set to 1.0 mm using the Odyssey infrared image system (LI-COR Biosciences, Lincoln, NE).
5
Quantifying OXPHOS Protein Levels in Caco-2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fourteen-days differentiated Caco-2 cells were washed with ice-cold HBSS, lysed, and sonicated in 50 mM TRIS-HCl (pH 7.4) supplemented with 1% Triton (Sigma Aldrich) and 10% protease inhibitor (P8340, Sigma Aldrich). Experiments were performed in triplicate. Protein concentrations were determined with the Bio-Rad DC colorimetric protein assay (Bio-Rad laboratories) according to manufacturer's protocol. Protein samples were heated at 50°C for 5 min, separated (30 μg protein per sample per lane) by SDS-PAGE using 12% polyacrylamide gels and finally transferred to an Immobilon membrane (Millipore). Five individual proteins representing the five different OXPHOS complexes (NDUFB8 for Complex I; SDHB for Complex II; UQCRC2 for Complex III; COX II for Complex IV; ATP5A for Complex V) were detected (incubation overnight at 4°C) with total anti-OXPHOS human antibody cocktail (1:1,000, Ab110411, Abcam). Anti-β-actin antibody (1:1,000, Ab8227) was used as a loading control. Bound primary antibodies were visualized using IRDye 800CW goat anti-mouse IgG secondary antibody (1:10,000; LI-COR Biosciences Inc., Lincoln, NE, USA). Blots were scanned with LI-COR's Odyssey Infrared Image System.
6
Western Blot Analysis of MBD3 Knockdown
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blots to confirm MBD3 knockdown were performed as previously described [22 (link)]. In brief, cells expressing shRNAs for 7 days were acquired by cell sorting, lysed and proteins resolved by SDS–PAGE. Blotting was performed by using a wet-tank blotting system (BioRad, Hercules, CA, USA), and membranes were stained with primary antibodies anti-MBD3 (1:5000), anti-histone H3 (1:2000) or anti-β-actin (1:5000). Membranes were stained with secondary antibodies IRDye® 800CW (LI-COR Biosciences, Lincoln, NE, USA) or IRDye® 680RD (LI-COR Biosciences, Lincoln, NE, USA) (1:15,000) and imaged by using the Odyssey Infrared Image System (LI-COR Biosciences, Lincoln, NE, USA).
7
Western Blotting of Acetylated NF-κB
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Western Blotting, 100 mg of brain tissue was homogenized in RIPA (150 mM NaCl, 10 mM Tris (pH 7.2), 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA, supplemented with protease inhibitor mixture and phosphatase inhibitor) and centrifuged at 10,000× g for 20 min at 4 °C. Then, 20–40 µg of proteins from total homogenates were resolved in 12% acrylamide gels for SDS-PAGE and transferred to Immobilon-FL membranes (Millipore, Burlington, MA, USA). NFκB (p65) (acetyl K310) (cat# ab19870) were diluted 1:1000 (v:v) in PBS with 1% BSA and 0.1% Tween 20. Mouse anti-GAPDH used at 1:20,000 (v:v) (clone 6C5 #MAB374 from Sigma-Aldrich) or Revert™ 700 Total Protein Stain for Western Blot Normalization (LI-COR) was used as a loading control. Secondary antibodies were diluted 1:20,000 (v:v) in 1% BSA and 0.1% Tween 20. Proteins were detected using the Odyssey Infrared Image System (LI-COR).
8
Western Blot Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were separated by 12% SDS-PAGE and then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were incubated with anti-HA mAb, anti-Flag mAb, and anti-M mAb, respectively. After the membranes were rinsed with PBST, each membrane was treated with DyLight 800-Goat Anti-Mouse IgG (H + L) as the secondary antibody. The proteins were visualized by scanning the membranes with a LI-COR Odyssey infrared image system (LI-COR Biosciences, Lincoln, NE, USA).
9
Western Blot Analysis of Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intact cells grown as for the transport assay were washed with 20 mM Tris-HCl, pH 7.5; 10 μl of cells at A600 = 15 were diluted to 40 μl, then sonicated in an ice-cold water bath (Branson 2510) for 5 min 20 μl of the whole cell lysate (equivalent to 7 μg protein as judged by protein assay) was loaded onto SDS-16%PAGE. The gel was transferred onto PVDF membrane by the Trans-Blot Turbo transfer system (Bio-Rad Laboratories) at 1.3 A, 25 V for 20 min. The blocked PVDF membrane by 3% BSA was then reacted with the penta-His antibody (Invitrogen), and washed in 20 mM Tris-HCl, pH 7.5, and 500 mM NaCl for 30 min, and further probed by the secondary Alexa-fluor 680 antibody (Invitrogen) diluted in 3% BSA, 0.1% Tween-20, and 0.01% SDS for 1 h. After washing with 20 mM Tris-HCl, pH 7.5, and 500 mM NaCl for 30 min, the membrane was scanned by LI-COR Odyssey Infrared Image system at 700 nm. Molecular weight markers were indicated (Precision plus protein WesternC standards, Bio-Rad Laboratories).
10
Western Blot Analysis of Hippocampal Proteins
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!