Cell samples were lysed for 2h at 4°C in lysis buffer containing 0.01M Tris-HCl (pH 7.4), 0.15M KCl, 0.1M NaF, 0.002M EDTA, 0.012M β-mercaptoethanol, 0.5% Nonidet P-40 and a cocktail of protease inhibitors (leupeptin 0.01mg/mL; 0.001M Na3VO4; Pefabloc 0.3mg/mL; 0.01μM okadaic acid). Protein were solubilized in sample buffer, heated at 95°C for 1 min, separated by SDS-PAGE on 10% polyacrylamide gel and transferred on a 0.2 μm nitrocellulose membrane. Primary antibodies raised in different species and secondary antibodies coupled with different fluorochromes, were sequentially combined to specifically label one marker in green (800 Li-Cor), the other in red (680 Li-Cor). The Odyssey™ imaging system (LI-COR Biotechnology, Lincoln, NE, USA) and the Azure c500 imaging system (Azure Biosystems, Dublin, CA, USA) was used to quantify the signals. Antibodies used are listed in Supplementary Table 2 .
Azure c500 imaging system
Manufactured by Azure Biosystems
Sourced in United States
The Azure c500 imaging system is a versatile and compact imager designed for a range of molecular biology applications. It captures high-quality images of various fluorescent and chemiluminescent samples, including gels, blots, and microplates. The system features a high-resolution camera, flexible light sources, and user-friendly software for image acquisition and analysis.
Automatically generated - may contain errors
Lab products found in correlation
5 bromo 4 chloro 3 indolyl phosphate bcip, by Merck Group (2 mentions)
Whatman, by Cytiva (2 mentions)
Odyssey imaging system, by LI COR (1 mentions)
β actin antibody, by Proteintech (1 mentions)
Brd4 13 440, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ecl chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Precast protein gel, by Bio-Rad (1 mentions)
Tris hcl gradient gel, by Bio-Rad (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Image studio lite version 5, by LI COR (1 mentions)
Odyssey 3, by LI COR (1 mentions)
Newblot pvdf stripping buffer, by LI COR (1 mentions)
Phosphatase inhibitor, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Beyotime (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Ap conjugated secondary antibodies, by Merck Group (1 mentions)
11 protocols using azure c500 imaging system
1
Immunoblotting for Protein Quantification
2
ARV-Induced Protein Expression in HepG2 Cells
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HepG2 cells were treated with 0.5 μM ARV for 48 h and cells were lysed in modified RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% v/v NP40, 0.5% w/v deoxycholate, 0.1% w/v SDS, 10% v/v glycerol, 10 mM NaF, 0.4 mM EDTA) with protease inhibitors. Cell lysates were centrifuged at 4 °C for 10 min at 10,000 g. The supernatant was collected to which Laemmli sample buffer containing SDS and β-mercaptoethanol was added. Samples were denatured by heating at 95 °C for 10 min. Subsequently, samples were separated on polyacrylamide gels and transferred to PVDF membrane, and probed with primary antibodies BRD4 (13440), c-Myc (5605), Bcl-2 (3498), Survivin (2808) from Cell Signaling Technology, and β-actin antibody (66009) from Proteintech. HRP-conjugated secondary antibodies were used along with enhanced chemiluminescence substrate. Images were obtained with the Azure C500 imaging system and quantified using ImageJ 1.8.0 software.
3
Western Blot Analysis of Protein Signaling
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Proteins were extracted from mouse liver tissue using RIPA lysis buffer that was suitably combined with a mixture of protease inhibitors and phosphatase inhibitors. Protein concentrations in the supernatant were quantified by using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then wet electrotransferred to polyvinylidene fluoride (PVDF) transfer membranes for 90 min. Then, 5% skim milk in TBST (20 mM Tris pH 7.5, 150 mM NaCl, and 0.1% Tween 20) was used to block the PVDF membranes at room temperature for 2 h. Subsequently, the membranes were incubated with primary antibodies against ERK, JNK, p38, I-κB and NF-κB and phosphorylated ERK, JNK, p38, I-κB and NF-κB overnight at 4°C. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000) for 2 h at room temperature. Finally, an ECL chemiluminescence substrate (Thermo Scientific, USA) was used to develop the immunoreactivity of the membranes. Protein-antibody complexes were detected using an Azure c500 imaging system (Azure Biosystems, USA) and normalized using GAPDH as an internal control. Two duplicates were performed for each condition.
4
Zymographic Analysis of MMP-1 Activity
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MMP-1 activity was analyzed by performing zymography on conditioned media obtained from the basal chamber of hiPSC-RPE cultures on transwells in accordance with a previously described protocol74 (link). Briefly, conditioned media samples were electrophoresed on 10% polyacrylamide gels containing 1 mg/ml collagen (Sigma-Aldrich). Subsequently, the polyacrylamide gels were incubated in 2.5% Triton-X-100 in 1X PBS for a period of 2 h. After three additional rinses, the gels were incubated in prewarmed MMP-1 incubation buffer (50 mM Tris-HCl, pH, 7.4, 5 mM CaCl2, 0.001% NaN3, 0.005% Triton-X-100, pH 7.75) for 36 h at 37 °C. Following a brief rinse in destaining solution (1:3:6 acetic acid:methanol:water), the gel was stained with 0.2% Coomassie blue solution for 2–3 h at room temperature. Finally, the gels were rinsed again in destaining solution and imaged on Azure C500 imaging system (Azure Biosystems, Dublin, CA, USA). Of note, quantitative analyses were carried out using Image Studio Lite Version 5.2 and Microsoft Excel.
5
Western Blot Protein Analysis
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Cells were grown to 70–80% confluency and directly lysed with RIPA buffer. Lysates were cleared by centrifugation (14,000 rpm, 10mins, at 4 °C) and quantified by DC Protein Assay Kit (Bio-Rad, Hercules, CA, US, 5,000,111) using the manufacturer's microplate assay protocol. Generally, 15–30 μg protein was resolved in 4–15% Precast Protein Gels (Bio-Rad, Hercules, CA, US, 4,561,086), and transferred onto nitrocellulose membranes. The membranes were blocked with 5% dry milk in Tris-Buffered Saline-Tween-20 (TBS-T) for 1hr then incubated overnight with primary antibodies (Supplementary Table 1) in 5% bovine serum albumin in TBS-T. After washing and incubating with the appropriate secondary antibody, protein signals were detected with Immobilon Western Chemiluminescent Horseradish Peroxidase (HRP) Substrate Kit (Millipore, Burlington, MA, US, WBKLS0500) using Azure C500 imaging system (Azure Biosystems, Dublin, CA, US). Signal intensities of select proteins were quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, US).
6
Western Blotting of hiPSC-RPE Proteins
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hiPSC-RPE cells were lysed and an equivalent microgram load of protein from untreated and treated samples were resolved on 4–20% Tris-HCl gradient gels (Bio-Rad) and transferred onto low fluorescence polyvinylidene difluoride (PVDF) membranes (Bio-Rad) as previously described34 (link) and probed with the following primary antibodies: RHO, ACTN, or CTSD (1:500, Santa Cruz Biotechnology). Secondary antibodies utilized were near-Infrared fluorescent (1:10,000, Licor Biosciences) or peroxidase-conjugated secondary antibodies (1:10,000, Azure Biosystems). The blots were either imaged on Odyssey Infrared Imager (Licor Biosciences) or developed using the Radiance Plus Chemiluminescence Kit and imaged on Azure C500 imaging system (Azure Biosystems). Of note, prior to reprobing with a different antibody, the PVDF membrane was stripped using NewBlot PVDF stripping buffer (Licor Biosciences) for 60 min at room temperature. Quantitative analysis of all the western blotting data was carried out using the image acquisition software (Licor Odyssey 3.0 and/or Image Studio Lite version 5.2) and Microsoft Excel.
7
Extraction and Analysis of α-Synuclein Protein
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For protein expression analysis of α-syn, the protein was extracted from the midbrain with RIPA buffer containing protease inhibitors, phosphatase inhibitors, and phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China). The tissue was then mechanically homogenized, followed by incubation on ice for 2 h, sonication for 3 min, and centrifuged at 12,000 rpm at 4°C for 30 min to take the supernatant and quantify.
For each sample, 100 μg of protein was loaded into 10% gels without denaturalization, and electrophoresis was performed at 60 V for 1 h to concentrate and 110 V for 1 h to separate at 4°C. Then, the proteins were transferred to PVDF membranes at 250 mA for 1 h at 4°C. Membranes were blocked with 5% skim milk for 1 h, washed with TBST (containing 0.1% Tween-20), and incubated at 4°C with anti-α-synuclein antibody (1:2,000) and anti-β-actin antibody (1:3,000) overnight. The next day, the membranes were washed and incubated with goat anti-mouse IgG antibody (1:500) for 1 h at RT, rewashed, and exposed with ECL (Millipore, Bedford, MA, United States). The bands were visualized using the Azure (c500) imaging system (Azure, CA, United States), and the Western blot results were analyzed by ImageJ software.
For each sample, 100 μg of protein was loaded into 10% gels without denaturalization, and electrophoresis was performed at 60 V for 1 h to concentrate and 110 V for 1 h to separate at 4°C. Then, the proteins were transferred to PVDF membranes at 250 mA for 1 h at 4°C. Membranes were blocked with 5% skim milk for 1 h, washed with TBST (containing 0.1% Tween-20), and incubated at 4°C with anti-α-synuclein antibody (1:2,000) and anti-β-actin antibody (1:3,000) overnight. The next day, the membranes were washed and incubated with goat anti-mouse IgG antibody (1:500) for 1 h at RT, rewashed, and exposed with ECL (Millipore, Bedford, MA, United States). The bands were visualized using the Azure (c500) imaging system (Azure, CA, United States), and the Western blot results were analyzed by ImageJ software.
8
Protein Expression Analysis by Western Blot
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Cell samples were lysed for 2h at 4°C in lysis buffer containing 0.01M Tris-HCl (pH 7.4), 0.15M KCl, 0.1M NaF, 0.002M EDTA, 0.012M β-mercaptoethanol, 0.5% Nonidet P-40 and a cocktail of protease inhibitors (leupeptin 0.01 mg/ml; 0.001M Na3VO4; Pefabloc 0.3 mg/ml; 0.01μM okadaic acid). Proteins were denatured in sample buffer, heated at 95°C for 1 min, separated by SDS-PAGE on 10% polyacrylamide gel and transferred on a 0.2 μm nitrocellulose membrane. Primary antibodies raised in different species and secondary antibodies coupled with different fluorochromes, were sequentially combined to specifically label one marker in green (800 Li-Cor), the other in red (680 Li-Cor). The Azure c500 imaging system (Azure Biosystems, Dublin, CA, USA) was used to quantify the signals by immunofluorescence detection. List of antibodies used is given in Supplementary Table 2 .
9
Western Blot Protein Analysis Protocol
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Cells were harvested and lysed in RIPA lysis buffer (50 mmol/L Tris–HCl, pH7.5, 150 mmol/L NaCl, 1% NP40), and lysates were cleared by centrifugation at 16,000 ×g for 5 min at 4 °C. Total cell extracts were subject to SDS-PAGE and transferred to nitrocellulose membranes (10,401,196; Whatman, Maidstone, UK). After blocking with 5% nonfat dry milk in TBST for 1 h at room temperature (RT), membranes were incubated with the indicated primary antibodies at 4 °C overnight and then the corresponding alkaline phosphatase (AP)-conjugated secondary antibodies (Sigma) for 1 h at RT. After three washes with TBST, the blots were reacted with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) (Sigma). After three washes with TBST, the membranes were reacted with an ECL sensitive kit (B500023; Proteintech, Rosemont, IL, USA) and developed by using an Azure c500 imaging system (Azure Biosystems, Dublin, CA, USA).
10
Protease Profiling of Tenocyte Supernatants
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Cell supernatants were evaluated for the presence and relative amounts of 35 proteases using the Proteome Profiler Human Protease Array Kit (R&D Systems) according to the manufacturer’s protocol. Equal volumes of supernatants from tenocytes stimulated 1 µg/ml recombinant human S100A8 (Abcam), S100A9 (Abcam), 10 ng/ml IL-1ß for 24 hours were pooled from three donors and applied to the respective array membrane. Samples were then mixed with a combination of biotinylated detection antibodies and incubated overnight at 4 °C. The membranes were then subject to a series of washes before addition of diluted solution of horseradish peroxidase-conjugated streptavidin at room temperature for 30 minutes. Visualization of protease expression was carried out by chemiluminescence and signal intensity was quantified using an Azure c500 imaging system (Azure Biosystems). Relative optical densities of immunoreactive bands were determined using Image Studio Lite software (Li-Cor Biosciences).
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