An EMSA was performed to verify binding between TFs and cis‐acting elements from the target gene promoters. The TF genes were cloned into expression vectors containing the tag proteins (His, GST, and MBP) and transferred into competent cells of the E. coli strain Rosetta. Expression of TFs was induced with IPTG, and the proteins were purified using protein purification resins and eluted with tag protein elution buffer. The eluted proteins and biotin end‐labelled duplex DNA probe were mixed with EMSA binding buffer, incubated at 25 °C for 30 min to allow the TF proteins to interact with the probe, and then resolved by native gel electrophoresis using 0.5×TBE, Then, the protein–DNA complexes were transferred rapidly from native gel to a positive nylon membrane using E‐Blotter. The membrane was then transferred to a UV cross‐linked apparatus for 6 min, and then blocked with 15 mL of 1×blocking buffer for 30 min. After the blocking buffer was discarded, the membrane was incubated with 15 mL of 1:750 diluted streptavidin‐HRP prepared in blocking buffer at room temperature for 30 min, washed for 5 min with wash buffer (four times with shaking), and then incubated with 1.6 mL chemiluminescence substrates buffer. Finally, the membrane was visualized using a Tanon‐5200 Chemiluminescent Imaging System (Tanon Science & Technology, Shanghai, China).
Tanon 5200 chemiluminescent imaging system
Manufactured by Shanghai Tanon Science & Technology
Sourced in China, United States
The Tanon-5200 Chemiluminescent Imaging System is a laboratory equipment designed for the detection and analysis of chemiluminescent signals. It utilizes a sensitive camera and advanced optics to capture and quantify chemiluminescent signals generated in various biological and biochemical assays.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Ab136119, by Abcam (3 mentions)
Ab199428, by Merck Group (2 mentions)
Ab95997, by Santa Cruz Biotechnology (2 mentions)
Ab17721, by Abcam (2 mentions)
Sc542, by Cell Signaling Technology (2 mentions)
H3k4me2, by PTM Biolabs (2 mentions)
Ub h2a, by Cell Signaling Technology (2 mentions)
Gapdh, by Bioworld Technology (2 mentions)
Ap0063, by Shanghai Tanon Science & Technology (2 mentions)
Protease inhibitor mixture, by Roche (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
As014, by ABclonal (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Protein loading buffer, by Transgene (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Bca protein assay reagent kit, by Beyotime (1 mentions)
48 protocols using tanon 5200 chemiluminescent imaging system
1
Protein-DNA Binding Verification via EMSA
2
ELK1 Protein DNA Binding Assay
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Different DNA probes were end labeled with 6-carboxy-fluorescein (FAM), as shown in Supplemental Table 3 . 50 nM FAM-hm-DNA (1 pmole per lane) was preincubated with 500 ng ELK1 protein (Sino Biological) in reaction buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 8% glycerol, and 1 mM DTT) for 20 minutes on ice. The samples were then subjected to 10% polyacrylamide gel electrophoresis and run in 0.5 × Tris-borate-EDTA buffer at 100 V for 1 hour at 4°C. Images were visualized using a Tanon-5200 chemiluminescent imaging system (Tanon Science & Technology Co., Ltd.).
3
Protein Interaction Assay Using Split Luciferase
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The split luciferase complementation assays were performed as described previously (Fujikawa and Kato, 2007; Chen et al., 2008) . The full-length CDS of ICE1 was cloned into the pCAMBIA1300-nLUC vector under the control of the 35S promoter. Full-length CDS of NPR1 and TGA3 were cloned into the pCAMBIA1300-cLUC vectors. After co-expression in Nicotiana benthamiana leaves for 48 h，the leaves were sprayed with 1 mM D-luciferin (0.01% Triton X-100) solution and then incubated for 5 min in the dark before detecting.
Luciferase intensity was visualized on Tanon-5200 Chemiluminescent Imaging System (Tanon Science&Technology). The primers used for vector construction are listed in Supplemental Table S1.
Luciferase intensity was visualized on Tanon-5200 Chemiluminescent Imaging System (Tanon Science&Technology). The primers used for vector construction are listed in Supplemental Table S1.
4
Western Blot Analysis of STAT3 Phosphorylation
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Total protein samples from epididymal WAT and subcutaneous WAT were isolated by treating the tissues with RIPA lysis buffer supplemented with phosphatase inhibitor and protease inhibitor cocktail (Applygen, Beijing, China). The BCA Protein Assay Reagent Kit (Beyotime Biotechnology, China) quantified the protein concentrations. Proteins (10 μg) were separated using 4%-12% SDS-PAGE gels, and then transferred onto PDVF membranes (MilliporeImmobilon-PSQ, ISEQ00010, USA). After being blocked in blocking reagent (Beyotime Biotechnology, China), the membranes were incubated overnight at 4°C with primary antibodies, including Rabbit anti-β actin antibody (CST, 4970, USA), antisignal transducer and activator of transcription 3 (STAT3) antibody (CST, 4904, USA), anti-phospho-STAT3 (Tyr705) (CST, 9145, USA) antibody. Then the membranes were incubated with the corresponding secondary antibodies (CST, 7074, USA) for 1 h at room temperature. The Tanon-5200 Chemiluminescent Imaging System (Tanon Science & Technology, Beijing, China) was used for signal detection. The protein expression levels were quantified by the Image J software (version 1.48, National Institutes of Health, Bethesda, MD, USA).
5
Western Blot Analysis of eNOS and nNOS
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The tissues were homogenized in an ice-cold RIPA buffer, and then the tissue lysates were centrifuged at 12000×g and 4°C for 15 min. The supernatants were collected, and the total protein concentrations were measured using BCA assay. An aliquot of 30 µg of each protein sample was separated by electrophoresis on an 8% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane using a wet transfer system (LIUYI Biotech Co. Ltd, China). Nonspecific binding sites were blocked by 5% nonfat dry milk in 0.1% Tween-20 phosphate-buffered saline and then incubated overnight at 4°C with protein primary antibodies against eNOS (1 : 500; BD Transduction Laboratories Co. Ltd, USA), nNOS (1 : 500, BD Transduction Laboratories Co. Ltd, USA), and beta-actin (1 : 1000; 4A Biotech Co. Ltd, China). After washing, the membranes were incubated in horseradish peroxidase-conjugated goat antirabbit IgG (4A Biotech Co. Ltd, China) at room temperature for 2 h. The reaction antigen was detected with an enhanced chemiluminescence detection system (Millipore, USA) and visualized on Tanon-5200 Chemiluminescent Imaging System (Tanon Science & Technology Co. Ltd, China). Bands were quantitated by densitometry using ImageJ analyzer software.
6
Western Blot Analysis of Nrf2 Pathway
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Tissue protein from colons was collected by homogenization in a 5-fold volume of ice-cold RIPA lysis buffer and centrifugation at 3000 rpm for 30 min. The BCA method was utilized to determine the protein concentration in supernatants with bovine serum albumin as a standard. Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Millipore, USA). The membrane was blocked for 1 h and subsequently incubated with primary antibodies Kelch-like ECH-associated protein 1(Keap1, 1:1000), Nuclear factor erythroid 2-related factor 2 (Nrf2,1:1000), or Heme Oxygenase-1 (HO-1, 1:1000) at 4℃ overnight. The corresponding horseradish-peroxidase-conjugated secondary antibody (Cell Signaling Technology) was used to incubate with the membrane for another 1 h. The membrane was reacted with western blot ECL detection reagents and visualized with Tanon-5200 Chemiluminescent Imaging System (Tanon Science & Technology). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.
Results were quali ed with the ImageJ software.
Results were quali ed with the ImageJ software.
7
Cell Lysis and Protein Analysis
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The cells were treated as described above and were collected by centrifugation with 500g for 5 min, then washed twice in cold PBS and lysed with RIPA Lysis Buffer (Beyotime, Shanghai, China). The lysates were quantified using a BCA protein quantification kit (Yeasen). The proteins were separated by SDS-PAGE, immunoblotted, and visualized using the Tanon 5200 Chemiluminescent Imaging System (Tanon Science & Technology), then the images were cropped by Corel DRAW X3 SP2 software (https://www.coreldraw.com/en/pages/coreldraw-x3/ ).
8
HBV Capsid Detection in HepG2 Cells
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At 72 h after transfection, HepG2 cells were lysed with core lysis buffer for 30 min at room temperature. The cell lysates were centrifugated. Then the supernatants were collected and added with DNA buffer. The mixed supernatants were fractionated by electrophoresis through nondenaturing 1.8% agarose gels and transferred to a nitrocellulose membrane by dipping in with TNE buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1 mM EDTA). The membrane was hybridized with anti-HBc after fixing and blocking. Second antibody was HRP-linked anti-mouse IgG and HBV capsids were visualized by Tanon-5200 Chemiluminescent Imaging System (Tanon Science & Technology).
9
Protein Extraction and Western Blot Analysis
10
Mitochondrial Protein Expression Analysis
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Total cell extracts or nuclear extracts were separated by SDS-PAGE and transferred to PVDF membranes. The following antibodies were used for immunoblot analysis: anti-HK2, anti-PFKM, anti-LDHa1, anti-PDK2, anti-PDHa1, anti-ATP5B, anti-IDH2, anti-HIF1α, anti-MnSOD, anti-Cu/ZnSOD, anti-Catalase, anti-GR, anti-GRX1, anti-GPX1, anti-PRX3 and anti-TRX2 antibodies and secondary antibody were purchased from Proteintech. anti-MnSOD (acetyl K68) antibody was purchased from Abcam. Anti-β-actin, anti-ComplexV, anti-Lc3B, anti-MFN1, and anti-Fis1 antibodies were from Cell Signaling Technology. Protein expression is visualized on Tanon-5200 Chemi-luminescent Imaging System (Tanon Science Technology).
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