Aliquots of 50 µg of protein were loaded onto 10% sodium dodecyl sulphate-polyacrylamide gels for electrophoresis, and transferred to PVDF membranes. Membranes were blocked with skimmed milk for 1.5 h, and then primary antibodies against PGK2, Cfap69, Lin28b, Igf2bp1, GAPDH or β-actin were incubated overnight at 4 °C. The membranes were rinsed for three times and then incubated with the corresponding secondary antibodies. Finally, the blots were detected using an Odyssey infra-red imaging system (Thermo Fisher Scientific, Waltham, MA). The housekeeping protein GAPDH or β-actin was used to standardize the protein density.
Odyssey infrared imaging system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Odyssey Infrared Imaging System is a high-performance lab equipment designed for quantitative Western blot and 2D gel imaging. It utilizes infrared detection technology to provide sensitive and accurate protein detection and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Bis tris gel, by Bio-Rad (2 mentions)
Chemiluminescence kit, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Primary antibodies against hif 1α, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Runx2, by Cell Signaling Technology (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Fluorescently labelled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
8 protocols using odyssey infrared imaging system
1
Protein Expression Analysis by Western Blot
2
Western Blot Analysis of HIF-1α
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Cells were lysed in radioimmunoprecipitation assay buffer (RIPA) supplemented with phosphatase and protease inhibitors (Millipore, USA), as described previously [42 (link)]. Total protein was determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, USA). Equal amounts of proteins were electrophoresed on a 10% Bis-Tris gel (Bio-Rad, USA) and subsequently transferred to polyvinylidene fluoride membranes (Millipore, USA). The membranes were then incubated with primary antibodies against HIF-1α (Cell Signaling, USA) and then incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling, USA). The blots were detected using a chemiluminescence kit (Thermo Fisher Scientific, USA) and visualized using the Odyssey Infrared Imaging System.
3
Protein Extraction and Western Blot Analysis
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Cells were recovered from the constructs at the indicated time and dissolved in lysis buffer (Thermo Fisher Scientific, United States), supplemented with inhibitors of phosphatases and proteases (Millipore, United States) as described previously (Bougault et al., 2009 (link); Ge et al., 2019 (link)). BCA Protein Assay Kit (Pierce, United States) was used to determine the concentration of protein. Equal amount of protein was electrophoresed on a 10% Bis–Tris gel (Bio-Rad, United States) before transferring onto a nitrocellulose membrane. The membranes were then incubated with the following antibodies: SOX9 (Millipore, United States), RUNX2 (Cell signaling, United States), and GAPDH (Cell signaling, United States). All primary antibodies were applied at 1:1,000 dilution. Blots were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell signaling, United States). The immune complexes were detected using a chemiluminescence kit (Thermo Fisher Scientific, United States) and visualized via the Odyssey Infrared Imaging System.
4
Western Blot Analysis of Protein Samples
5
Western Blot Protein Detection Protocol
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Protein samples were resolved in SDS-PAGE and transferred to nitrocellulose membranes, blocked (30 min) in TBST buffer (50 mM Tris/HCl pH 7.5, 0.15 M NaCl, 0.1% (v/v) Tween) with 5% (w/v) dry milk, then incubated in TBST buffer with 5% (w/v) dry milk and 0.5-1 μg/ml antibody (2 h, room temperature (RT) or overnight, 4°C). Protein was detected using either horseradish peroxidase-conjugated secondary antibodies and the enhanced chemiluminescence reagent (Amersham Pharmacia Biotech), or fluorescently labelled secondary antibodies (Invitrogen) and the Odyssey infrared imaging system.
6
Comprehensive Protein Analysis Techniques
7
Protein Expression and Immunoblotting
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Seeds or seedlings were ground directly in 1× or 2× Laemmli loading buffer, respectively, microfuged 10 min at 4 °C to pellet debris, then boiled 5 min prior to fractionation by SDS-PAGE (10% polyacrylamide). Proteins were transferred to nitrocellulose filters, as described in [32 (link)]. Filters were blocked with Casein blocking buffer (LI-COR Biosciences, Lincoln, NE, USA), then co-incubated with anti-GFP mAb (1:10,000, UBPBio, Aurora, CO, USA) and anti-RGA pAb (1:2000, AS11 1630, Agrisera, Vännäs, Sweden) primary antibodies, followed by anti-mouse and anti-rabbit secondary IRDye 800 conjugated IgGs, and visualized using the 800 channel of the Licor Odyssey Infrared Imaging System or the iBright FL1500 Imaging System (Invitrogen, ThermoFisher Scientific, Waltham, MA USA). Filters were subsequently probed with anti-ABI5pAb (1:10,000, Ab98831, AbCam, Cambridge, UK) and anti-actin mAb (A0480, Sigma, St. Louis, MO, USA), followed by anti-mouse secondary IRDye 800 conjugated IgGs (LI-COR Biosciences, Lincoln, NE, USA).
8
Western Blot Analysis of Protein Expression
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Total protein of heart tissues and cultured NRVMs was extracted and protein concentration was measured with a Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). We separated 50 ug of samples through SDS-PAGE (8% polyacrylamide gel) and then transferred them to polyvinylidene difluoride (PVDF) membranes. The membranes were subjected to antigen blockage for 1 h at room temperature using 5% non-fat milk and incubated with diluted primary antibodies overnight at 4°C. Then, the membranes were washed with TBS (Tris-buffered saline) 3 times and incubated with IR Dye 800-conjugated secondary antibodies (Invitrogen) at room temperature for 2 h. After washing with TBS, the Odyssey Infrared Imaging System was utilized to visualize the protein-binding in membranes, and intensities of the bands were analyzed using Quantity One software. GAPDH was used as an internal control.
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