For SDS-PAGE analysis, precast 4–20% Bis-Tris gradient gels (Genscript, Picastaway, USA) in a Tris-MOPS running buffer was used. The molecular weight marker was Pierce™ Unstained MW marker (Thermo Scientific). Maximum volume of 20 µL was loaded in the wells on the SDS-PAGE gel. Solubilized extracts were mixed 14:5:1 with a 4xSDS sample buffer (50 mM Tris (VWR, Leuven, Belgium) pH 6.8, 2% SDS, 10% glycerol (VWR), 0.02% bromophenol blue (Sigma-Aldrich), 12.4 mM ethylenediaminetetraAcetic acid (EDTA, Carl Roth, Karlsruhe, Germany)) and 1 M dithiothreitol (DTT, Thermo Scientific), corresponding to a final DTT concentration of 50 mM. The samples incubated at 95 °C for 5 min to denature proteins. Running time for the gel was 45 min at 140 V. To visualize proteins, the gel was stained with Coomassie Brilliant Blue G250 (29 mM Coomassie Brilliant Blue G-250 (Sigma-Aldrich), 45% Ethanol (VWR), 10% Acetic acid (Fisher Scientific, Loughborough, UK)). Destaining of the gel was performed overnight with destain solution (8% Ethanol, 5% Acetic acid). Imaging of the gel was done using a ChemDoc MP Imaging System (Bio-Rad, Hercules, USA).
Chemdoc mp imaging system
Manufactured by Bio-Rad
Sourced in United States
The ChemDoc MP Imaging System is a versatile, high-performance imaging device designed for a wide range of applications in life science research. It captures images of stained gels, blots, and other samples using various detection modes, including chemiluminescence, fluorescence, and colorimetric. The system features a sensitive CCD camera, adjustable excitation and emission filters, and a user-friendly software interface for image acquisition and analysis.
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Lab products found in correlation
Typhoon fla 9500 laser scanner, by GE Healthcare (2 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Acetic acid, by Thermo Fisher Scientific (1 mentions)
Bis tris gradient gels, by GenScript (1 mentions)
Coomassie brilliant blue g 250, by Merck Group (1 mentions)
Bromophenol blue, by Merck Group (1 mentions)
Glycerol, by Avantor (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Carl Roth (1 mentions)
P p70s6k thr389, by Cell Signaling Technology (1 mentions)
β actin ac 15, by Merck Group (1 mentions)
P mlc ser19, by Cell Signaling Technology (1 mentions)
Liberase tl, by Roche (1 mentions)
Lamin b1, by Proteintech (1 mentions)
Rabbit polyclonal antibody against irf3, by Proteintech (1 mentions)
Enhanced chemiluminescence ecl, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Proteintech (1 mentions)
Powerpac, by Bio-Rad (1 mentions)
19 protocols using chemdoc mp imaging system
1
SDS-PAGE Protein Analysis Protocol
2
Protein Expression Analysis in Collagen Gels
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Cells were extracted from collagen gels by the action of Liberase TL (Roche) for 1 h at 37 °C. After having been lysed and dosed, protein samples were separated using SDS-PAGE and transferred onto nitrocellulose membranes in a transfer buffer (25 mM Tris, 200 mM glycine, Ethanol 20%). The blots were blocked with 5% low-fat milk in Tris-buffer saline (65 mM Tris pH 7.4, 150 mM NaCl) at room temperature for 1 h. They were incubated overnight with primary antibodies at 4 °C: p-p44/42 MAPK (Thr202/Tyr204) (E10, 1:1000), p-MLC Ser19 (#3671, 1:500), p-P70S6K Thr389 (1A5, 1:500, Cell signaling), β-actin (AC-15, 1:1000, Sigma-Aldrich), HSC-70 antibody (B-6, 1:5000, Santa Cruz Biotechnology). After being washed with TBS, the blots were incubated for 1 h with an HRP secondary antibody (1:1000) in 5% low-fat milk in TBS at room temperature. The blots were then washed with TBS. Immunocomplexes were visualized with a ChemDoc MP Imaging System (Bio-Rad) after a chemiluminescent reaction using the Ommobilon Western Chemiluminescent HRP substrate (Merck Millipore).
3
RT-PCR Amplification and Gel Analysis
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RT-PCR was used for cDNA amplification. cDNA templates from first-strand synthesis (1.5 ng) were amplified using 400 nM forward and reverse primers (supplemental Table S2 ), 1X PCR TaqFast MasterMix with dye, and DEPC-treated water to a final volume of 50 μl. Samples were incubated in thermal cycler programmed to the following: Step 1/initial denaturation: 3 min at 94°C. Step 2/denaturation: 30 s at 94°C. Step 3/annealing: 30 s at primer specific temperature. Step 4/extension: 1 min at 72°C. Step 5: Repeat steps 2–4 for primer-specific number of cycles. Step 6/final extension: 10 min at 72°C. Step 7/final holding: 4°C until use or storage at –20°C. Amplification products were analyzed by gel electrophoresis with 1% agarose gel in 0.5X TAE and stained with GelRed (1:10,000, v/v). Gels were imaged with BioRad ChemDoc MP Imaging System (Nucleic Acid GelRed setting).
4
Cerium-Mediated DNAzyme Cleavage Assay
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The DNAzyme complex was annealed in buffer C (25 mM NaCl, 50 mM MES, pH 6.0) with 1 μM FAM-labeled substrate strand and 2 μM enzyme. At room temperature, 10 μM Ce3+ was added to initiate the cleavage reaction. At each designated time point, a 2.5 μl aliquot was mixed with 14 μl urea (8 M) to quench the reaction. The cleavage products were separated using 15% denaturing polyacrylamide gel electrophoresis (dPAGE) and analyzed by a ChemDoc MP imaging system (Bio-Rad).
5
Western Blot Analysis of Protein Expression
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Cell samples were lysed with a radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) and subjected to SDS-PAGE. Proteins in the gel were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore) and blocked with 5% non-fat milk in TBST buffer. The blots on the membrane were detected by primary antibodies and corresponding horseradish peroxidase-conjugated secondary antibodies (ProteinTech, Wuhan, China). These antibodies were diluted in a TBST buffer containing 5% non-fat milk, and the membrane was washed with a TBST buffer containing 0.1% Tween-20. Finally, the membrane was visualized by enhanced chemiluminescence (ECL) (Millipore), and protein bands were visualized by image using the ChemDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). A mouse monoclonal antibody against GAPDH (ABclonal, Wuhan, China), a rabbit monoclonal antibody against IFITM3 (CST, Danvers, MA, USA), and a rabbit polyclonal antibody against IRF3 (Proteintech) and Lamin B1 (Proteintech) were purchased from indicated companies.
6
Quantitative PCR Protocol for Amplicon Analysis
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Quantification of non-specific and specific amplicons was performed by quantitative PCR (Roche LightCycler 480 II, Switzerland). The PCR mixture for non-specific amplification consists of 400 U/mL Nt. BstNBI, 80 U/mL Vent® (exo-) DNA polymerase, 1 × Thermopol Buffer, 2 mM MgSO4, 100 mM template, 250 μM dNTP, 0.5 × NEB buffer, 1 × SYBR Green Ⅰ, and a series of concentration of TMAC ranging from 0 mM to 300 mM with a total volume of 50 μL. For specific amplification, 200 mM blockers were included in the PCR mixture. The amplification program included 55°C for 90 min, and the frequency of fluorescence signal collection was 1 min. The qPCR products were further characterized by 12% native polyacrylamide gel electrophoresis. The polyacrylamide gel mixture included 4 mL 30% w/v acrylamide/methylene bisacrylamide (29:1), 2 mL 5 × TBE, 20 μL N,N,N′,N′-tetramethylethylenediamine, 200 μL 10% w/v ammonium persulfate, and distilled water to a final volume of 10 mL. The analytes were incubated in 1 × TBE at 90 mV for 100 min on the Universal Power Supply (Bio-Rad PowerPac™, USA). After that, gels were stained with 4S Red Plus Nucleic Acid Stain and visualized with a ChemDoc™ MP Imaging System (Bio-Rad, USA).
7
Protein Separation and Visualization
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One dimensional (1D) SDS-PAGE analysis was performed with a SureLock Xcell system (Thermo Fisher Scientific). Protein samples were mixed with 25% NuPAGE Sample Buffer and 10% Reducing Agent and heated at 70°C for 10 min. About 20 μg of proteins were loaded into wells of 4%-12% acrylamide NuPAGE Bis-Tris gels. The proteins were separated at 200 V for 35 mins in MES SDS running buffer. Gels were stained with Bio-Safe Coomassie Blue G250 and scanned in a ChemDoc MP imaging system (Bio-Rad).
For 2D gel electrophoresis, 200-μg samples of proteins were first cleaned with the ReadyPrep 2D Cleanup Kit (Bio-Rad) or by precipitation with acetone and then dissolved in 185 μL of ReadyPrep rehydration buffer. Proteins were separated first by isoelectric focusing in 11-cm IPG (immobilized pH gradient) strips (pH 3 to 10; Bio-Rad). Proteins were then separated in the second dimension by SDS PAGE with 8-16% gradient Criterion Tris-glycine polyacrylamide gels and Tris-glycine SDS buffer (Bio-Rad). Gels were stained with Bio-Safe Coomassie Blue G250 and scanned in a ChemDoc MP imaging system.
For 2D gel electrophoresis, 200-μg samples of proteins were first cleaned with the ReadyPrep 2D Cleanup Kit (Bio-Rad) or by precipitation with acetone and then dissolved in 185 μL of ReadyPrep rehydration buffer. Proteins were separated first by isoelectric focusing in 11-cm IPG (immobilized pH gradient) strips (pH 3 to 10; Bio-Rad). Proteins were then separated in the second dimension by SDS PAGE with 8-16% gradient Criterion Tris-glycine polyacrylamide gels and Tris-glycine SDS buffer (Bio-Rad). Gels were stained with Bio-Safe Coomassie Blue G250 and scanned in a ChemDoc MP imaging system.
8
Neutrophil DNA Degradation Analysis
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DNA electrophoresis was performed to determine the effects of ArtinM treatment on neutrophils DNA degradation. The analysis was performed using a kit, the Wizard SV genomic DNA purification system (Promega Corporation, USA). Briefly, after 24 and 48 h of incubation, 4 × 106 cells were washed twice with PBS and lysed, and genomic DNA was isolated. The extracted DNA was quantified using the NanoVue Plus spectrophotometer (GE Healthcare, USA). A sample of 1 μg of DNA was analyzed using 1.5% agarose gel electrophoresis and stained with ethidium bromide. The DNA was then visualized under UV light on ChemDoc MP Imaging System and photographed using ImageLab software v.4.0 (both from Bio-Rad Laboratories, USA). Regarding the analysis of DNA fragmentation, the band area was quantified using ImageJ Software, and represented graphically in pixels2.
9
Western Blot Analysis of Protein Markers
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Brain samples were collected and proteins were obtained with lysis buffer (Thermo Scientific) supplemented with protease inhibitors (Sigma‐Aldrich). After electrophoresis, the proteins were transferred to PVDF (polyvinylidene fluoride) membranes. The membranes were blocked by incubation with 5% dry milk for 2 h at room temperature and then incubated with primary antibodies against ferritin (diluted 1:1000, Abcam, ab75973), alpha smooth muscle actin (α‐sma, diluted 1:1000, Abcam, ab5694), TAGLN / transgelin (Sm22α, diluted 1:1000, Abcam, ab89989), and GAPDH (diluted 1:2000, zsbio, TA‐08) at 4°C overnight. After thorough washing, the membranes were incubated with HRP‐conjugated IgG (diluted 1:2000, Proteintech) for 2 h at room temperature. The protein bands were visualized using ECL kits (Thermo Scientific) and imaged with a ChemDoc MP imaging system (Bio‐Rad). Images of the blots were analyzed using ImageJ software (NIH).
10
Gel-Based DNAzyme Activity Assay
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For a typical gel-based activity assay, a final concentration of 0.85 μM FAM-labeled substrate strand and 2 μM enzyme strand were added to 50 mM Tris buffer (pH 7.5) containing 100 mM monovalent metal ion. The DNAzyme complex was formed by incubating at 80°C for 2 min and then slowly cooling to 4°C. At room temperature, target metal ions were added to initiate the cleavage reaction. The reaction was quenched by transferring 2.5 μl sample to 14 μl urea (8 M) at designate time points. The cleaved products were separated using 15% denaturing polyacrylamide (PAGE) gel and analyzed by a Bio-Rad ChemDoc MP imaging system.
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