Whole‐cell extracts were obtained by lysis in TBSN buffer [20 mm Tris (pH 8.0), 150 mm NaCl, 0.5% Nonidet P‐40, 5 mm EGTA, 1.5 mm EDTA, 0.5 mm Na3VO4, and 20 mm p‐nitrophenyl phosphate] supplemented with protease inhibitors. Western blotting was performed with the indicated antibodies, and protein levels were developed using ECL Western Blot Substrate (NCM Biotech, Suzhou, China). Azure 600 Gel Imaging System, together with the azure capture software (1.6.3.1211, Azure Biosystems, Dublin, CA, USA) and azurespot (2.2.167) from Azure Biosystems were used to capture western blot bands.
Azurespot
Manufactured by Azure Biosystems
Sourced in United States
The AzureSpot is a versatile and compact imaging system designed for capturing high-quality images of a variety of samples. It features a sensitive CCD camera, efficient LED illumination, and flexible sample positioning options. The AzureSpot is a reliable tool for tasks such as gel documentation and basic fluorescence detection.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo transfer system, by Bio-Rad (4 mentions)
Sapphire biomolecular imager, by Azure Biosystems (4 mentions)
Gel doc xr system, by Bio-Rad (3 mentions)
Image lab, by Azure Biosystems (2 mentions)
Mini protean tgx gel, by Bio-Rad (2 mentions)
Antirabbit or antimouse igg secondary antibody, by Bioss Antibodies (2 mentions)
Rna oligos, by Integrated DNA Technologies (1 mentions)
N n methylene bis acrylamide, by Bio-Rad (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Prism software, by GraphPad (1 mentions)
Rabbit anti sars cov 2 s, by Sino Biological (1 mentions)
Mouse anti tubulin, by Thermo Fisher Scientific (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Complete protease inhibitor tablet, by Roche (1 mentions)
Ecl detection system, by Promega (1 mentions)
Azure sapphire biomolecular imager, by Azure Biosystems (1 mentions)
11 protocols using azurespot
1
Western Blot Protein Extraction and Analysis
2
EMSA Analysis of PRPF39 Binding
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EMSA was performed using IRDye-700 labeled 10 nt poly(A) and GC (GGCCCCCCGG) RNA oligos (Integrated DNA Technologies). PRPF39 was added to the RNA sample (10 nM) in binding buffer (50 mM Tris pH 7, 150 mM NaCl, 20% glycerol, 3 mM MgCl2, 1 mM DTT) to final concentrations of 270, 617, 964, 1311, 1658, 2005, 2352, and 2700 nM. The concentration range for PRPF39 NTD and CTD were 10, 30, 90, 270, 810, 2430, and 7290 nM. All reactions had a final volume of 5 µL including murine RNA inhibitor (New England Biolabs) and were incubated at 30°C for 15 min. The EMSA samples were analyzed on a nondenaturing PAGE (5% acrylamide; 37.5:1 acrylamide: N,N-methylene-bis-acrylamide [BioRad]) with 0.2× TBE buffer (26 mM Tris pH 7.5, 9 mM boric acid, 0.5 mM EDTA) and 2.5% glycerol (Liu et al. 2016 (link)). The gels were prerun at 100 V for 40 min before loading sample and subsequently electrophoresed at 100 V for 65 min. The RNA was visualized using an Odyssey infrared imaging system (LiCor). The band intensities were measured using AzureSpot (Azure Biosystems) analysis software. After background subtraction, the fraction of RNA bound was calculated, fitted with the Hill equation, and graphed using Prism software (GraphPad).
3
SDS-PAGE Protein Analysis Protocol
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Cell and virus lysates in lysis buffer with 6x SDS-PAGE sample loading buffer (600 mM Tris-HCL pH6.8, 30% glycerol, 12% SDS, 20mM DTT, 0.03% bromophenol blue) were heated at 95°C for 5 min. Samples were analyzed on 10% 1.5mm Tris-glycine gels using a BioRad Trans-Blot Turbo Transfer system according to manufacturer’s instructions. Proteins were detected with primary (see Table S2 ) and secondary antibodies. Protein bands were visualized using chemiluminescence with either a Gel Doc XR+ system (BioRad) or Sapphire Biomolecular Imager (Azure Biosystems) and analyzed with either Image Lab version 6.0.1 or AzureSpot (Azure Biosystems).
4
Western Blot Analysis of NF-κB, STAT1, and GAPDH
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Twenty-μg of protein was run in gel electrophoresis by using 4–20% Mini-PROTEAN® TGX™ Gel (Bio-Rad, CA, USA), and transferred onto a nitrocellulose membrane. The membranes were blocked for one hour at room temperature and incubated overnight at 4 °C with primary antibodies against Nfkb p65, Stat1, and Gapdh (Abcam, MA, USA) at a:1000 concentration. Following three times washing step using phosphate-buffered saline with Tween-20, the membranes were incubated with the antirabbit or antimouse IgG secondary antibody in a concentration of 1:10,000 (Bioss Antibodies, MA, USA) for 1 h at room temperature. Clarity™ Western ECL Substrate was used to detect protein bands. Gapdh was used as the loading control, and three replicates of experiments were performed to obtain statistical significance. The detected protein bands were quantified using AzureSpot (Azure Biosystems, CA, USA).
5
SDS-PAGE and Immunoblotting Analysis
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Cell and virus lysates in lysis buffer with 6× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (600 mM Tris-HCl [pH 6.8], 30% glycerol, 12% SDS, 20 mM dithiothreitol [DTT], 0.03% bromophenol blue) were heated at 95°C for 5 min. Samples were analyzed on 10% 1.5-mm Tris-glycine gels using a Bio-Rad Trans-Blot Turbo Transfer system according to the manufacturer’s instructions. Proteins were detected with primary antibodies (see Table S3 ) and secondary antibodies. Protein bands were visualized using chemiluminescence with either a Gel Doc XR+ system (Bio-Rad) or Sapphire biomolecular imager (Azure Biosystems) and analyzed with either Image Lab version 6.0.1 or AzureSpot (Azure Biosystems).
6
SDS-PAGE Protein Analysis Protocol
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Cell and virus lysates were treated with 6x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer [600 mM Tris-HCl (pH 6.8), 30% glycerol, 12% SDS, 20 mM dithiothreitol, 0.03% bromophenol blue] and heated at 95 °C for 5 min. Samples were analyzed on 4 to 15% Tris-glycine gels using a Bio-Rad Trans-Blot Turbo Transfer system according to the manufacturer’s instructions. Proteins were detected with primary and secondary antibodies. Protein bands were visualized using chemiluminescence with Gel Doc XR+ system (Bio-Rad) and analyzed with Image Lab version 6.0.1 or AzureSpot (Azure Biosystems).
7
AICD Densitometry Analysis of FAD Mutants
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AICD densitometry was carried out using Azurespot (Azure Biosystems). A standard curve established from a concentration range of C100-FLAG protein was used to calculate AICD-FLAG product concentration from the enzyme reaction mixtures. Unpaired two-tailed T-tests were used to compare measurements from FAD-mutant samples to those of WT samples.
8
SARS-CoV-2 Spike Protein Detection
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Cells were lysed in lysis buffer (10 mM iodoacetamide [Sigma-Aldrich], Complete protease inhibitor tablets [Roche], 300 mM sodium chloride, 50 mM Tris-HCl [pH 7.5], and 0.5% Triton X-100 [Sigma-Aldrich]). Virus was pelleted through 20% sucrose and resuspended in lysis buffer. Cell and virus lysates were heated to 95 °C for 5 min. Samples were analyzed on 10% 1.5 mm Tris-glycine gels, followed by transfer to PVDF membrane (BioRad) using a BioRad Trans-Blot Turbo Transfer system according to manufacturer's instructions. Blots were probed with the following antibodies: rabbit anti-SARS-CoV-2 S (Sino Biological; #40589), rabbit anti-SARS-CoV-2 S2 (Sino Biological; #40590), human anti-HIV immune globulin (HIV-Ig; NIH AIDS Reagent Program), and mouse anti-tubulin (Invitrogen; #62204). Secondary antibodies conjugated with horseradish peroxidase were used for chemiluminescent detection for SARS-CoV-2 S and other proteins were detected using near-IR fluorescently labeled secondary antibodies (Azure Biosystems 650 and 800; AC2165 and AC2135). Protein bands were visualized using a Sapphire Biomolecular Imager (Azure Biosystems) and analyzed with AzureSpot (Azure Biosystems).
9
Protein Expression Analysis in Rat Tissues
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Subsequently, 40 μg protein from 4 to 5 rats in each group was run in gel electrophoresis by using 4–20% Mini-PROTEAN® TGX™ Gel (Bio-Rad, CA, USA), followed by protein transfer onto nitrocellulose membranes. After blocking for 1 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies against Chp1, Serpina3 (1:1000, ABclonal technology, MA, USA), C3, or Vcl (1:1000, both from Santa Cruz, TX, USA), separately. The membranes were thoroughly washed with phosphate-buffered saline with Tween-20 and then incubated with the antirabbit or antimouse IgG secondary antibody (1:10000, Bioss Antibodies, MA, USA) for 1 h at room temperature. Clarity™ Western ECL Substrate was used to detect protein bands. Vinculin (Vcl) was used as the loading control, and one rat sample from the 6 M-CTL group was included in each gel as a reference for between-gel comparison. The detected protein bands were quantified using AzureSpot (Azure Biosystems, CA, USA).
10
Western Blot Protein Detection and Quantification
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Proteins were size‐fractionated by SDS–PAGE and transferred to nitrocellulose or polyvinylidene difluoride membranes by electroblotting. The nitrocellulose membranes were blocked with 5% nonfat dry milk or 5% BSA in TBST (TBS containing 0.1% Tween 20) for 60 min at room temperature and subsequently incubated with the primary antibody diluted in blocking buffer for 16 h at 4°C. After extensive washing with TBST, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibody for 60 min at room temperature. Following washing with TBST, the antigen was detected with the enhanced chemiluminescence (ECL) detection system (Promega) as specified by the manufacturer. In addition, immunoblots with fluorescently labeled secondary antibodies were imaged by an Azure Sapphire Biomolecular Imager (Azure Biosystems, USA). For quantification of Western blots, Image Studio lite (version 3.1) and Fiji were used for X‐ray films and AzureSpot (version 2.0) was used for the Sapphire Biomolecular Imager (Azure Biosystems, USA).
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