Leukocytes were isolated from SGs and spleens as described previously (Stacey et al., 2011 (link)). CD4+ T cells were purified via magnetic separation using a MagniSort Mouse CD4 Positive Selection Kit (Thermo Fisher Scientific). Cell lysates were generated from equal numbers of cells using NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) supplemented with 100 mM dithiothreitol (Sigma-Aldrich). Samples were loaded onto 4–12% NuPAGE Bis-Tris Gels (Thermo Fisher Scientific) after boiling and transferred to a PVDF membrane using an XCell II Blot Module (Thermo Fisher Scientific). Blots were probed with anti-arginase-1 (rabbit polyclonal, Thermo Fisher Scientific) and developed using anti-rabbit IgG–HRP (Bio-Rad). Band intensity was determined using a G:BOX Gel Imaging System (Syngene). Blots were then stripped using Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific) and probed again with anti-actin (rabbit polyclonal, Abcam).
G box gel imaging system
Manufactured by Syngene
Sourced in United Kingdom, United States
The G:BOX is a gel imaging system designed for capturing high-quality images of electrophoresis gels, such as those used in DNA, RNA, and protein analysis. It features a large imaging area, sensitive camera, and versatile illumination options to capture clear, detailed images of your samples.
Automatically generated - may contain errors
Lab products found in correlation
Ecl system, by Cytiva (5 mentions)
Hybond, by Cytiva (5 mentions)
Peroxidase conjugated secondary antibody, by Cell Signaling Technology (4 mentions)
Hybond ecl membrane, by Cytiva (4 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (4 mentions)
Amersham ecl prime, by Cytiva (4 mentions)
Nitrocellulose blotting membrane, by GE Healthcare (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Chef dr 3, by Bio-Rad (3 mentions)
Incert agarose, by Lonza (3 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase linked secondary antibody, by Cell Signaling Technology (2 mentions)
Anti gapdh antibody, by Cell Signaling Technology (2 mentions)
Anti β actin antibody, by Cell Signaling Technology (2 mentions)
Anti claudin 5 antibody, by Abcam (2 mentions)
Anti ve cadherin antibody, by Santa Cruz Biotechnology (2 mentions)
Anti zo 1 antibody, by Cell Signaling Technology (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Cell fractionation kit, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
38 protocols using g box gel imaging system
1
Purification and Western Blotting of CD4+ T Cells
2
Quantifying Cardiac Marker Proteins
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Hearts were homogenized in homogenization buffer containing 150 mM NaCl, 50 mM Tris–HCl, 50 mM NaF, 2% Triton X-100, 0.1% SDS, supplemented with phosphatase and proteases inhibitor cocktail set III (Calbiochem). Homogenates were then loaded onto polyacrylamide gels for SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked with 5% milk and probed with primary antibodies against atrial natriuretic factor (ANP; Millipore AB2232, 1:1000), brain natriuretic peptide (BNP; Abcam ab239510, 1:1000), and calcipressin-1 (ThermoFisher 14869-1-AP, 1:1000). Equal protein loading was verified by re-probing for GAPDH (Abcam ab8245). Bands were detected by chemiluminescent signals using the enhanced chemiluminescence method (SuperSignal West Dura Extended Duration Substrate, ThermoScientific, USA) and visualized with a G:BOX gel imaging system (SynGene, Cambridge, United Kingdom). Band intensity was measured using ImageJ software (NIH, Bethesda, USA). For each gel, the signal intensity was averaged over the control samples. Then, the signal intensity in all lanes was normalized to this average. This procedure was repeated on all technical replicates and the normalized signal intensity was averaged for each sample, followed by averaging over experimental groups.
3
Western Blot Analysis of Inflammatory Signaling
4
Western Blot Antibody Profiling
5
Mitochondrial Isolation and Protein Analysis
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Mitochondria were isolated from H9C2 cells using a mitochondrial isolation kit (Thermo Scientific) according to the manufacturer's protocol. Western blots were performed as described previously [19 (link)–21 (link)]. The membranes were incubated with appropriate primary antibodies respectively, including anti-PTEN, anti-p-Akt, anti-Akt, anti-Bim, anti-p-Bad, anti-Bad (Cell Signaling Technology, Inc, Danvers, MA), respectively, followed by incubation with peroxidase-conjugated second antibodies (Cell Signaling Technology, Inc.) and examination with the ECL system (Amersham Pharmacia, Piscataway, NJ). The signals were quantified using a G: Box gel imaging system by Syngene (Syngene, USA, Frederick, MD).
6
Western Blot Analysis of Liver Proteins
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Total protein of 100 mg snap-frozen liver tissues was extracted by RIPA lysis buffer and protein concentration was determined by BCA assay kit (Solarbio, Beijing, China) based on the manufacturer's protocols. 40 μg protein was loaded in 10% SDS-PAGE gel and transferred to PVDF membranes. Rabbit anti-IκBα (#4812, 1 : 1000, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-NF-κB P65 (#8242, 1 : 1000, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-CYP2E1 (ab28146, 1 : 2000, Abcam, Cambridge, UK), anti-Histone H3 (#4499, 1 : 2000, Cell Signaling Technology, Inc., Danvers, MA, USA), and anti-GAPDH (ab9484, 1 : 2500, Abcam, Cambridge, UK) antibodies were used and goat anti-rabbit IgG H&L (HRP) (ab205718, 1 : 5000, Abcam, Cambridge, UK) as the secondary antibody. The membranes were visualized by ECL reagents, and the protein bands were detected by G:BOX gel imaging system (Syngene, Cambridge, UK).
7
Western Blot Protein Extraction Protocol
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Protein extraction of cultured RPCs was carried out using a RIPA lysis buffer (Pierce, United States) complemented with a Protease Inhibitor Cocktail (Roche, Germany). Insoluble material was removed by centrifugation at 12,000 × g for 10 min at 4°C. Protein concentrations were determined using the BCA method (Pierce) and adjusted in line. A total of 20–40 μg of each protein was subjected to electrophoresis on 10–12% SDS polyacrylamide gels (SDS-PAGE) and transferred to PVDF membranes. Membranes were blocked for 1 h in 5% non-fat dry milk in Tris-HCl buffer containing 0.05% Tween-20 (TBST), after which they were incubated in primary antibodies (showed in Supplementary Table 1 ) overnight at 4°C. After washing three times in TBST, membranes were then incubated in an HRP-conjugated secondary antibody (showed in Supplementary Table 1 ) for 2 h at room temperature. After being thoroughly rinsed, immunoreactive bands were detected using an enhanced chemiluminescent (ECL) substrate (Pierce) and exposed to a Fuji X-ray film (Japan). The bands were collected with a G: box gel imaging system (Syngene, United Kingdom) and analyzed with the ImageJ software (NIH). The housekeeping β-actin was used as the internal control to normalize the levels of target proteins.
8
Western Blotting Protein Separation
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Western blotting was performed as described in Cowman et al. (2019) (link). Briefly, 30–40 µg of protein were separated on 10% SDS–PAGE gels. Proteins were transferred onto nitrocellulose membrane (0.2 µm), followed by primary and secondary antibody incubation. Signal was developed using Amersham ECL Prime Western blotting Detection reagent (GE Healthcare, Chicago, IL, USA), and images taken using a G:BOX gel imaging system (Syngene, Cambridge, UK).
9
Western Blot Analysis of Protein Expression
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20–30 µg protein were resolved on a 10% SDS-polyacrylamide gel, transferred onto a nitrocellulose membrane and probed with primary antibodies (anti-choline kinase alpha [Abcam; ab88053], anti-vinculin [Abcam; ab129002], anti-tubulin [Cell Signalling; 2128S], anti-E2F5 [Santa Cruz; sc-1083], anti-E2F1 [Cell Signalling; 3742], anti-α3+β1 [Abcam; ab217145], anti-HIF-1α [Protein Tech; 20960-1-AP], anti-HIF-2α [Bethyl Labs; A700–003]) at 4°C for 12 h. Membranes were incubated with anti-rabbit (1:10,000) horseradish peroxidase-linked secondary antibody (Cell Signalling) for 2 h at room temperature. Signal was developed using Amersham ECL Prime Western blotting Detection reagent (GE Healthcare, Chicago, IL, USA), and images taken using a G:BOX gel imaging system (Syngene, Cambridge, UK).
10
Western Blot Quantification Protocol
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Conducted as described in [30 ]. In brief, 20–40 μg of protein was resolved on a 10% SDS-PAGE gel and transferred to nitrocelloulose membrane (0.2 μM) before incubation in primary and secondary antibody. Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) was used for development of signal, and a G:BOX gel imaging system (Syngene, UK) used for detection. Western blot quantification was conducted with ImageJ 1.45 s open source software (National Institutes of Health, USA).
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