Ten micograms of protein from each sample (whole homogenate and SNPs) was electrophoretically resolved in 10% TGX StainFree FastCast Acrylamide Solutions (Bio-Rad) at 200 V for 1 h. After resolution, protein was semi-dry transferred onto 0.2 µm PVDF membranes (#1704272, Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad). After blocking membranes in 5% (w/v) BSA, primary antibodies were applied at 1:1000 overnight at 4°C. Proteins probed from the hippocampus were mTOR (#2972S, CST), p-mTOR (#2971S, CST), Synapsin I (#6710, CST), PSD-95 (#2507S, CST), GluA1 (#13185S, CST), p-GluA1 (#75574S, CST), p-Erk1/2 (extracellular signal regulator kinase; #9101S, CST), CREB (#4820S, CST), p-CREB (#9198S, CST), and GluN2b (UC Davis). Blots were subsequently washed in tris-buffered saline with 1% (v/v) Tween (TBST) before application of appropriate secondaries (horseradish peroxidase linked goat anti-mouse or goat anti-rabbit) at a 1:5000 concentration. To visualize bands, Luminata Crescendo or Classico was used depending on the abundance of protein (#WBLUR0500 and #WBLUC0500, Millipore Sigma). The G:BOX Chemi XRQ imaging system (Syngene) was used to capture chemiluminescent images, and Fiji (version 2.9.0) was used for quantification of bands. Ponceau staining was used for total protein normalization.
G box chemi xrq imaging system
Manufactured by Syngene
Sourced in United Kingdom
The G:BOX Chemi XRQ is a high-performance imaging system designed for capturing and analyzing chemiluminescent, fluorescent, and colorimetric signals. It features a high-resolution camera, multiple excitation and emission filters, and adjustable light sources to accommodate a variety of sample types and applications.
Automatically generated - may contain errors
Lab products found in correlation
Tgx stain free fastcast acrylamide solutions, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Pierce protein free blocking buffer, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Socs3, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl detection system, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Anti rabbit peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
5 protocols using g box chemi xrq imaging system
1
Protein Expression Profiling in Hippocampus
2
Quantitative Western Blotting of Midbrain Organoids
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At least five midbrain organoids from three different batches, at day 60 and day 90 timepoints, were lysed in Western ready-rapid protein extraction buffer (BioLegend), boiled and protein concentrations were determined using Pierce BCA protein assay (Thermo Scientific). Equal amount of denatured proteins were separated via SDS–polyacrylamide gel electrophoresis on a 4%–20% Mini-PROTEAN TGX precast protein gel (BioRad) and transferred to a polyvinylidene difluoride (PVDF) membrane (pore size, 0.45 mm). The membrane was blocked in Pierce Protein-Free blocking buffer (ThermoFisher Scientific) and probed overnight with an appropriate primary antibody (Table 1). The membrane was treated with a secondary anti-mouse or anti-rabbit, horseradish peroxidase-conjugated antibody (1:10,000). Western blots were developed using Pierce ECL Western Blotting Substrate (Thermo Scientific) and chemiluminescence images were obtained using G:Box Chemi XRQ imaging system (Syngene). Quantitative analysis of the western blotting data was carried out on three different blots using ImageJ software.
3
Western Blot Protein Analysis
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Tris-glycine SDS-PAGE was undertaken using an OmniPAGE VS10DSYS vertical electrophoresis system (OmniPAGE, Cleaver Scientific Ltd., Rugby, UK) to separate proteins before wet transfer to a PVDF membrane (EMD Millipore Corporation, Billerica, MA, USA).
Once complete, the membrane was incubated in blocking solution (5% low fat milk and 0.1% Tween 20 in TBS) for at least 1 hour at room temperature. The membrane was then probed with primary antibody (SOCS-4, R & D systems, Abingdon, UK; SOCS-3, Abcam, Cambridge, UK; GAPDH, Insight Biotechnology Ltd., Middlesex, UK) at 4 °C overnight, subjected to washes and incubated with a horseradish peroxidase (HRP) conjugated secondary antibody (Anti-mouse or Anti-goat, Sigma-Aldrich, Dorset, UK) for 2 hours before washing and protein band detection using a EZ-ECL Chemiluminescent Detection Kit (Biological industries, Kibbutz Beit-Haemek, Israel) and visualisation in a G:BOX Chemi XRQ imaging system (Syngene, Cambridge, UK).
Once complete, the membrane was incubated in blocking solution (5% low fat milk and 0.1% Tween 20 in TBS) for at least 1 hour at room temperature. The membrane was then probed with primary antibody (SOCS-4, R & D systems, Abingdon, UK; SOCS-3, Abcam, Cambridge, UK; GAPDH, Insight Biotechnology Ltd., Middlesex, UK) at 4 °C overnight, subjected to washes and incubated with a horseradish peroxidase (HRP) conjugated secondary antibody (Anti-mouse or Anti-goat, Sigma-Aldrich, Dorset, UK) for 2 hours before washing and protein band detection using a EZ-ECL Chemiluminescent Detection Kit (Biological industries, Kibbutz Beit-Haemek, Israel) and visualisation in a G:BOX Chemi XRQ imaging system (Syngene, Cambridge, UK).
4
Evaluating Molecular Biomarkers in Cell Lines
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30 × 104of DLD-1 cells were seeded into 100 cm2 Petri dishes and treated with the equivalent IC50 concentration of EVO and
p-coumaric acid. Following the incubation, the cells were washed with pre-cold PBS and lysed in RIPA buffer (Cell Signaling Technology, Beverly, MA, USA) with 1 mM of PMSF. The lysates were centrifuged at 14,000 g at 4°C for 15 min. The supernatant was used to determine the protein concentration, which was determined by the BCA method. Fifteen µg of the protein samples were separated by SDS-PAGE (4% stacking gel and 7.5% separating gel), blotted onto PVDF membrane, and incubated with polyclonal (rabbit) anti-APC, anti-TP53, anti-BRAF, anti-SMAD4, anti-KRAS antibodies (Bio-Rad Laboratories, Hercules, CA, USA) overnight at 4°C, and with goat anti-rabbit IgG secondary antibody for 1 h at RT. ECL detection systems (Thermo Scientific Co.) were used for detection. Gels were photographed using the Syngene GBOX Chemi XRQ imaging system (Frederick, MD, USA) and analyzed using the GeneSys software.
p-coumaric acid. Following the incubation, the cells were washed with pre-cold PBS and lysed in RIPA buffer (Cell Signaling Technology, Beverly, MA, USA) with 1 mM of PMSF. The lysates were centrifuged at 14,000 g at 4°C for 15 min. The supernatant was used to determine the protein concentration, which was determined by the BCA method. Fifteen µg of the protein samples were separated by SDS-PAGE (4% stacking gel and 7.5% separating gel), blotted onto PVDF membrane, and incubated with polyclonal (rabbit) anti-APC, anti-TP53, anti-BRAF, anti-SMAD4, anti-KRAS antibodies (Bio-Rad Laboratories, Hercules, CA, USA) overnight at 4°C, and with goat anti-rabbit IgG secondary antibody for 1 h at RT. ECL detection systems (Thermo Scientific Co.) were used for detection. Gels were photographed using the Syngene GBOX Chemi XRQ imaging system (Frederick, MD, USA) and analyzed using the GeneSys software.
5
Endotoxin-induced RhoA deamidation assay
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We treated 5 × 105 A549, MC-3T3-E1, B3Z, and polarized VA10 cells with 10, 100, or 1000 ng/mL of endotoxin-free recombinant DNT or its DNT-C1305S variant used as negative control. After 4 h of incubation at 37 °C, the cells were lysed with 50 mM Tris-HCl (pH 8.0), 200 mM DTT, 0.3% SDS, Complete mini inhibitors (1 tablet per 10 mL, Merck, Darmstadt, Germany), heated for 3 min at 99 °C and then treated with benzonase (Novagen, Madison, WI, USA) for 30 min at 4 °C. The lysates containing 10 µg of the total protein were separated by Tris-Tricine SDS-PAGE and transferred to nitrocellulose membranes. The deamidated RhoA bearing Glu63 was detected by the anti-RhoA63E antibody 6MmDNT22–2 at a 1:250 dilution (provided by Yasuhiko Horiguchi, Osaka University), while total RhoA was detected by the anti-RhoA antibody EP487Y at a 1:1000 dilution (Abcam, Cambridge, UK). Primary antibodies were then probed by peroxidase-conjugated anti-rabbit secondary antibody at a 1:5000 dilution (GE Healthcare BioSciences, Pittsburgh, PA, USA). After washing, the membranes were incubated with a SuperSignal Sensitivity Substrate kit (Thermo Fisher Scientific, Waltham, MA, USA) and the chemiluminescence signal was detected using a G:BOX Chemi XRQ imaging system (Syngene, Cambridge, UK).
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