Immunoblotting was carried out as previously described (1 ) and analyzed using either a G:BOX Chemi (Syngene, Hyarana, India) CCD camera with Genesnap software (Syngene) or a LI-COR Odyssey Fc (LI-COR, Nebraska, USA). Densitometry analysis was undertaken using ImageStudio software (Version 5.2.5, Li-COR). For complement studies, SDS-PAGE under non-reducing conditions was performed on patient/parental serum (diluted 1:125 in non-reducing buffer [PBS]) or affinity-purified factor H (diluted to 200 ng in non-reducing buffer), separated by electrophoresis on a 6% SDS-PAGE gel and transferred to nitrocellulose membranes for immunoblotting (antibodies in table S6 ). Blots were developed with Pierce ECL Western blotting substrate (ThermoFisher Scientific) and imaged on a LI-COR Odyssey Fc (LI-COR).
Odyssey fc
Manufactured by LI COR
Sourced in United States, Germany, Niger
The Odyssey Fc is a versatile imaging system designed for a wide range of fluorescence-based applications. It provides high-quality, quantitative imaging of fluorescent samples, including Western blots, gels, and microplates. The system's key features include a sensitive CCD camera, multiple excitation and emission filters, and advanced imaging software for data analysis and presentation.
Automatically generated - may contain errors
Lab products found in correlation
Image studio software, by LI COR (25 mentions)
Pvdf membrane, by Merck Group (19 mentions)
Odyssey blocking buffer, by LI COR (16 mentions)
Protease inhibitor cocktail, by Merck Group (10 mentions)
Nitrocellulose membrane, by Bio-Rad (9 mentions)
Protease inhibitor cocktail, by Roche (8 mentions)
Protran, by Cytiva (8 mentions)
Image studio lite, by LI COR (8 mentions)
Image studio, by LI COR (7 mentions)
Image studio lite software, by LI COR (7 mentions)
Image studio v5, by LI COR (7 mentions)
Turbo transfer system, by Bio-Rad (7 mentions)
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (6 mentions)
Image studio 4, by LI COR (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Revert total protein stain, by LI COR (6 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (5 mentions)
Donkey anti rabbit igg hrp, by Santa Cruz Biotechnology (5 mentions)
Irdye 800cw, by LI COR (5 mentions)
329 protocols using odyssey fc
1
Immunoblotting and Densitometry Analysis
2
Quantitative Protein Expression Analysis in Liver Tissue
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Briefly, liver tissues were dissected and lysed in RIPA lysate (CAT: P0013B) and centrifuged, and the supernatant collected. Bicinchoninic acid (BCA) protein concentration assay kit (CAT: P0012A) was used to determine protein concentration. Vertical electrophoresis separation was performed. The primary antibody incubated with a membrane at 4 °C, for 12–16 h. TBST (TBS + Tween) was washed 3 times for 15 min each. Then, the secondary antibody was incubated with a membrane at room temperature, for 2 h. After 3 washes of TBST, the membrane was irradiated with a chemiluminescent solution. The developed strips were scanned with Odyssey Fc (Odyssey Fc, LI-COR Biosciences, 4647 Superior Street, Lincoln, NE, USA) two-color infrared fluorescence imaging system, and the gray value of each strip was determined by analysis with Image J software. The ratio of the gray value of the obtained target strip to the gray value of the corresponding internal reference β-actin strip was used to determine the relative expression of the corresponding target protein.
3
In Vitro Ubiquitination Assay Protocols
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Multiturnover activity assays were carried out at 37 °C for the indicated times in 20 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM MgCl2, 2 mM DTT (1114GR005, BioFroxx), and 10 mM ATP (A7699, Sigma-Aldrich), containing E1 (0.1 μM), E2 (10 μM), E3 (5 μM), and ubiquitin (50–100 μM) (final concentration). The reactions were incubated at 37 °C and stopped by the addition of a reducing SDS sample buffer. For some assays, Cy3-labelled ubiquitin was used in 1:1 ratio with the unlabelled form9 (link). Following separation by SDS-PAGE reactions containing Cy3-labelled ubiquitin were imaged with an Odyssey® Fc (LI-COR Biosciences) using a 600 nm filter prior to Coomassie® Brilliant Blue R-250 (APA1092.0025, AppliChem) staining.
For single-turnover ubiquitin discharge assays, 15 μM of E2~Ub conjugate variants were incubated with 0.125–5 mMl -lysine (L5501, Sigma-Aldrich) and 0.25–1 μM of E3 ligase. All assays were incubated at 25 °C and then stopped by mixing with non-reducing 2× SDS sample buffer, which ensured that the unhydrolyzed thioester E2~Ub conjugate remained intact. Samples were resolved by SDS-PAGE and the reactions containing Cy3-labelled E2~Ub conjugate were imaged by an Odyssey® Fc (LI-COR Biosciences) using 600 nm filter prior to Coomassie® Brilliant Blue R-250 (APA1092.0025, AppliChem) staining.
For single-turnover ubiquitin discharge assays, 15 μM of E2~Ub conjugate variants were incubated with 0.125–5 mM
4
Protein Extraction and Western Blotting
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Cells were lysed in 1% NP-40, 50 mM Tris (pH 8.0) and 150 mM NaCl, supplemented with 1x Complete Protease Inhibitor Cocktail (11836170001; Roche, ON, Canada) and phosSTOP (04906837001; Roche). When required, subcellular fractionation was performed using a designated kit from Cell signaling (9038S) according to the manufacturer’s recommendations. The protein content was subsequently, adjusted using the bicinchoninic acid (BCA) assay. Samples were then separated on 4–12% or 12% Bis-Tris (Life Technologies) or 7% Tris-glycine gels (cast in-house). The proteins were then transferred to a 0.45 micron polyvinylidene fluoride membrane, blocked in 10% skimmed milk and probed overnight at 4°C with the respective antibody diluted in 5% skimmed milk. On the next day, the blots were incubated with HRP-conjugated anti-mouse (1:5000, 170–6516; BioRad, ON, Canada) or anti-rabbit (1:5000, 170–6515; BioRad) secondary antibodies and the ECL reagent (RPN2106; GE Healthcare). The signal was then visualized using a LI-COR Odyssey Fc digital imaging system (LI-COR Biosciences, NE, USA).
5
Quantifying Membrane Protein Expression
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Surface biotinylation samples were heated to 50 °C for 10 min before separation using 4% to 20% polyacrylamide gradient gels (Bio-Rad, Hercules, CA, USA). After separation, proteins were transferred to nitrocellulose membranes using Invitrogen's Power Blotter System. Blots were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature while rocking. After blocking, blots were incubated overnight at 4 °C with a mouse antibody against the α subunit of Na+/K+-ATPase (Abcam-ab7671; 1:2000) and with a mouse antibody against the His tag (Tetra·His Antibody, QIAGEN catalog no. 34670; 1:2000) in blocking solution on a rocker. The next day blots were washed with Tris-buffered saline (TBS) containing 0.1% Tween 20 and TBS before incubation with a horseradish peroxidase–conjugated goat antimouse secondary antibody at 1:10,000 (Thermo Fisher Scientific, catalog no. 31430) in 2.5% milk in TBS. After 1 h, blots were washed with TBS and incubated with SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific). Blots were visualized using a LI-COR Odyssey Fc (LI-COR, Lincoln, NE, USA), and bands were quantified using their Image Studio Lite Quantification Software.
6
Western Blot Analysis of Signaling Proteins
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Example 10
Lysis was performed using RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail (Roche). QuickStart Bradford protein assay (BioRad) was used to determine protein concentration with BSA as standard. Equal amounts of total protein were mixed with NuPAGE lithium dodecyl sulphate (LDS) and sample reducing buffer (Thermo Scientific), heated for 5 min at 95° C. and loaded into NuPAGE 10% or 12% Bis-Tris gels (Life Technologies) for electrophoresis. Separated proteins were transferred onto nitrocellulose membranes using iBlot 2 gel transfer device (Thermo Scientific). Blocking was performed with 5% milk or bovine serum albumin (BSA) in tris-buffered saline supplemented with 0.1% tween (TBST). AKT (4298S), ERK1/2 (4348S), p-AKT (9271S), and p-ERK (8544S) antibodies were from Cell Signaling Technology. RON antibody was made in-house and Actin-HRP (A3854) was from Sigma-Aldrich. Anti-rabbit (P0217) and anti-mouse (P0161) secondary antibodies were from Dako. The enhanced chemiluminescence (ECL) reagent used was SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, #34076). Imaging and acquisition was performed with Licor Odyssey Fc and Image Studio (version 3.1).
7
Protein Quantification by Western Blot
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Western blots were performed as described55 (link) and detected by LICOR –Odyssey-Fc imaging (LI-COR Biosciences, Lincoln, NE). Densitometry was conducted on exposures within the linear range.
8
Protein Extraction and Western Blot Analysis
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Cells were lysed with 50 mM Tris⋅HCl (pH 7.6) containing 1% NP40, 30 mM NaCl, 1 mM EDTA, 150 mM NaCl, and 1× proteinase inhibitor cocktail (cat. # 4693159001; Millipore Sigma) or RIPA buffer supplemented with EDTA-free protease inhibitor cocktail (11873580001; Sigma-Aldrich) and 1 mM PMSF. Protein concentrations were determined using Protein Assay Kit (5000002; BioRad). Lysates were resolved on 10% NUPAGE Bis-Tris gels (Invitrogen), transferred to polyvinylidene fluoride membranes, and incubated with the relevant antibodies. Stained membrane signals were visualized by using Hyglo HRP detection kit (cat. # E2500; Denville Scientific) and exposed to HyBlot CL film (cat. # E3012; Denville Scientific) by LICOR Odyssey Fc (LI-COR Biosciences). Protein levels were quantified using ImageJ (https://imagej.nih.gov/ij/ ). Details on antibodies used are listed in Table S8 .
9
Protein Expression Analysis of Aortic Samples
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Briefly, SDS-PAGE was used to resolve equal amounts of protein isolated from the aorta along with the molecular weight marker, transferred to PVDF membrane. Milk (5%) was used for blocking the membrane, followed by incubation with respective primary antibodies rabbit polyclonal to SM22-α (1:10,000, abcam, Cambridge, MA, USA), rabbit polyclonal to RUNX2 (1:1000, abcam, USA) and rabbit anti-β-actin (1:10,000, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. Next day incubated with donkey anti-rabbit-HRP IgG (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. Finally, bands were detected by chemiluminescence technique using LI-COR Odyssey Fc and the band intensity of target proteins was normalized to β-actin and calculated with Image J software version 1.44 p (NIH, Bethesda, MD, USA) [71 (link)].
10
Heme Abundance Quantification by Immunoblotting
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