Human CRC HT-29 and HCT116 cells were treated or not with the determined IC50 values of compounds (12a, 10a, 10b ) for indicated times (6, 12, 24 and 48h) and then harvested with trypsin. For total protein extraction, collected samples of each condition were washed with PBS. Then, the total cell pool was centrifuged at 200× g for 5 min at 4 °C and homogenized in RIPA lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, 0.1% Sodium Dodecyl Sulfate (SDS), 20 mg/mL of aprotinin) containing protease inhibitors according to the manufacturer’s instructions as previously described [30 (link)]. Proteins (60 µg) were separated on 12.5% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were probed with respective human antibodies against caspase-3, cleaved caspase-3, PARP and Akt, ERK, p38 MAP Kinases and its phosphorylated forms according to the manufacturer’s instructions. After incubation with appropriate secondary antibodies, blots were developed using the «Immobilon Western » substrate following the manufacturer’s protocol and G:BOX system (Syngene, Ozyme). Membranes were then reblotted with human anti-β-actin used as a loading control.
G box system
Manufactured by Syngene
Sourced in United Kingdom, United States, India, France
The G:Box system is a versatile imaging platform designed for a range of laboratory applications. It provides high-quality image capture and analysis capabilities for various sample types. The core function of the G:Box system is to enable researchers and scientists to document, analyze, and interpret their experimental results with precision and efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (4 mentions)
Genetools software, by Syngene (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
β actin, by Merck Group (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Ecl chemiluminescence, by Merck Group (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Westernbright sirius chemiluminescent detection kit, by Advansta (2 mentions)
Laemmli sample buffer 4x concentrate, by Bio-Rad (2 mentions)
Mouse anti actin, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Dcode apparatus, by Bio-Rad (2 mentions)
Genesys software, by Syngene (2 mentions)
Pvdf membrane, by Cytiva (2 mentions)
0.45 m pvdf membrane, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Anti mettl14, by Cell Signaling Technology (2 mentions)
72 protocols using g box system
1
Western Blot Analysis of Apoptotic and Signaling Pathways in CRC Cells
2
Protein Detection in N. benthamiana Leaves
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For each sample, three discs 1 cm in diameter were collected from agro-infiltrated N. benthamiana leaf patches, five days post infiltration (dpi), ground in 270 µL of phosphate buffered saline (pH 7.4) in the presence of protease inhibitors (cOmplete™: Roche Diagnostics GmbH, Mannheim, Germany), and mixed with 1 volume (w/v) of Laemmli buffer 2X [38 (link)]. Total soluble proteins were heat-denatured 10 min at 90 °C and clarified at 10,000 g for 5 min prior to separation on an 8% acrylamide SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Resolved proteins were electro-transferred onto Immobilon PVDF (Polyvinylidene Difluoride) membranes and incubated with commercial polyclonal anti-TagRFP antibodies (Evrogen, Moscow, Russia) at a 1:5000 dilution. The detection step was achieved with goat anti-rabbit antibodies conjugated to horseradish peroxydase at a 1:12,500 dilution (Life technologies, Thermo Fisher Scientific) and with the Lumi-Light chemiluminescence system (Roche Diagnostics GmbH). The membrane was then imaged with the G:Box system (Syngene, Cambridge, UK).
3
Quantification of Proteins in Villous Tissue
4
Generation of hCD5 Transgenic Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DNA sequence coding for the whole extracellular region of CD5 was amplified from the pHβAPRI-neo-CD5.P346stop construct [18] (link) and subcloned into XhoI-ApaI restricted pIgHSV40s vector which contained the SV40 promoter and the immunoglobulin μ heavy chain enhancer (Eμ). This vector, termed pIgHSV40shCD5, was injected into fertilized eggs from a CBAxC57Bl/6 mixed background. Founder mice were backcrossed for 10 generations into the C57Bl/6 background. Non-transgenic littermates were used in all experiments. Transgenic mice were identified by PCR analysis of genomic DNA samples from ear punch specimens in a GeneAmp PCR System 2700 termocycler (Applied Biosystems, USA). The cycling conditions were: 30 cycles of 5 min at 94°C, 1 min at 92°C, 1 min at 53°C, and 8 min at 72°C. The primers to detect the transgene were specific for the extracellular region of human CD5 (forward, 5′-GCTGTCCCAGTGCCACGAACTT-3′ ; reverse, 5′-GAAGCTCCTCTGTGTCCTCAT-3′ ). Internal control amplification primers (forward, 5′-TCACTCAAGGCAACCTTCCTGC-3′ ; reverse, 5′-CGACCTCATCTCTAACCATGAACAG-3′ ) specific for the invariant chain Ii of MHC class II (LIEX) were also included. The resulting PCR products (450 and 150 bp, respectively) were separated in a 2% agarose gel and visualized in a G:Box system (SynGene, UK).
5
Analyzing Proline Dehydrogenase Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Leaves (100–150 mg) were ground in a fine powder in liquid nitrogen. The frozen powder was then suspended into 2 volumes of extraction buffer containing 8 M urea, 5% (w/v) SDS, 40 mM Tris-HCl pH 6.8, 0.1 mM EDTA, and 0.4 mg ml–1 bromophenol blue, supplemented with 1× protease inhibitor cocktail (Merck). Samples were shaken three times for 5 min with 5 min intervals on ice. The samples were then centrifuged at 18 000 g for 10 min at 10 °C and the supernatants (30 µl) were loaded and separated onto a 8% polyacrylamide gel. The proteins were then transferred onto nitrocellulose membranes (Amersham Hybond-ECL® from GE Healthcare) and were immunoblotted using purified antibodies directed against either Arabidopsis recombinant ProDH1, ProDH2, P5CDH, or P5CS proteins. Antibody binding was detected using a secondary antibody conjugated with a horseradish peroxidase and the ECL prime system from GE Healthcare using the G:BOX® system (Syngene).
6
Western Blot Analysis of Wnt Pathway Proteins
7
Quantification of Placental Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein concentration in placental homogenates was determined using the Bradford assay (Bio-Rad). The placental homogenate (10 μg) was separated on 4%–20% precast linear gradient gels (Invitrogen). Membranes were incubated overnight at 4°C with primary antibody diluted in 1% non-fat milk (wt/vol) in TBST and detected using an appropriate peroxidase-conjugated secondary antibody. Products were visualized by ECL chemiluminescence (Millipore). Band intensities were measured using the G-box system (Syngene). Anti-β actin was purchased from Sigma-Aldrich, St. Louis, Mo. Target band densities were normalized using beta-actin to account for any variation in loading and transfer. Additional protein expression analysis was performed using automated capillary-based immunoassay (ProteinSimple, San Jose, CA, catalog #SM-W004-1, #PS-ST01, and #PN-009–050), as described previously (Castillo-Castrejon et al., 2021 (link)). Briefly, Jess plates were run according to the manufacturer’s instructions, with minor modification (200 V, 55 min separation time) with 0.1 mg/mL total protein concentration.
8
Oxidative Stress Protein Analysis in Whole Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysates were collected using SDS lysis buffer supplemented with protease inhibitor cocktail (Sigma-Aldrich, UK). Denatured samples were separated by gel electrophoresis, electro-transferred onto polyvinylidene difluoride membranes (Millipore, Sigma, USA) and then probed with primary and HRP-conjugated secondary antibodies (Millipore, Sigma, USA). Membranes were probed for HIF-1α (Abcam, UK), catalase (Calbiochem, UK), CuZnSOD (Abcam, UK), MnSOD (R&D Systems, Minneapolis), NQO1 (Santa Cruz, USA), HO-1 (BD Biosciences, USA), Bach1 (Santa Cruz, USA), metallothionein (MT1/2, Abcam, UK), ZnT1 (Abcam, UK) and β-actin (Sigma-Aldrich, UK). Membranes were subjected to development with enhanced chemiluminescence (Millipore, Sigma, USA) with images captured using a G:Box system (Syngene, UK). Immunoblot densitometry data were analyzed using ImageJ software (National Institute of Health, USA).
9
Immunoprecipitation of NRP1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with cold PBS and lysed in 50 mM Tris buffer (pH 7.4) containing 150 mM NaCl, 1% Triton X-100, 1 mM EDTA and protease and phosphatase inhibitors cocktail (Roche). For cytosol and membrane proteins extractions, cells were prepared using a subcellular fractionation kit (Thermo Scientific) with protease and phosphatase inhibitors according to the manufacturer’s instructions.
For immunoprecipitation, proteins were prepared using lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10% glycerol, and protease and phosphatase inhibitors. Five hundred micrograms of total protein extract was incubated with 1 µg of anti-NRP1 antibody or control IgG overnight at 4 °C. Complexes were pulled down using Bio-Adembeads Protein A/G magnetic beads (Ademtech), washed with lysis buffer and analysed by SDS-PAGE. Immunostaining was visualised using the GBox system (Syngene). Band intensities were quantified using the Multi Gauge v3.0 software (Fujifilm).
For immunoprecipitation, proteins were prepared using lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10% glycerol, and protease and phosphatase inhibitors. Five hundred micrograms of total protein extract was incubated with 1 µg of anti-NRP1 antibody or control IgG overnight at 4 °C. Complexes were pulled down using Bio-Adembeads Protein A/G magnetic beads (Ademtech), washed with lysis buffer and analysed by SDS-PAGE. Immunostaining was visualised using the GBox system (Syngene). Band intensities were quantified using the Multi Gauge v3.0 software (Fujifilm).
10
Western Blot Protein Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates were prepared with a modified NP-40 lysis buffer that contained the following: 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5% NP-40, 10% glycerol, complete protease inhibitor tablets (Roche, Indianapolis, IN), 1 mM PMSF, 50 mM β-glycerol phosphate, 10 mM p-nitrophenyl phosphate, 2.5 mM sodium pyrophosphate, 100 nM okadaic acid, 10 mM sodium fluoride, 1 mM sodium orthovanadate, and 10 μM phenylarsine oxide.
Protein concentrations were quantified using the bicinchoninic acid assay (BCA) method (Bio-Rad, Hercules, CA). Routinely, 25 μg of protein was run on either 4% to 12% Tris-HCl gradient gels (Bio-Rad) or 4% to 20% Bis-Tris Criterion gradient gels (Bio-Rad). Proteins were transferred to either 0.2-μm polyvinyl difluoride (PVDF) or 0.45-μm nitrocellulose membranes. Membranes were blocked and incubated with primary antibodies according to standard Western blot analysis protocols. Subsequent to primary antibody binding, membranes were incubated with the corresponding HRP-conjugated secondary antibody. The proteins were detected by chemiluminescence (GE Healthcare, Piscataway, NJ) with exposure to X-ray or detected in the G-Box system (Syngene, Frederick, MD). Band intensities were analyzed with Gene Tools software (Syngene). Raw volume with background subtracted was normalized to GAPDH, with vehicle-treated cells set as 100%.
Protein concentrations were quantified using the bicinchoninic acid assay (BCA) method (Bio-Rad, Hercules, CA). Routinely, 25 μg of protein was run on either 4% to 12% Tris-HCl gradient gels (Bio-Rad) or 4% to 20% Bis-Tris Criterion gradient gels (Bio-Rad). Proteins were transferred to either 0.2-μm polyvinyl difluoride (PVDF) or 0.45-μm nitrocellulose membranes. Membranes were blocked and incubated with primary antibodies according to standard Western blot analysis protocols. Subsequent to primary antibody binding, membranes were incubated with the corresponding HRP-conjugated secondary antibody. The proteins were detected by chemiluminescence (GE Healthcare, Piscataway, NJ) with exposure to X-ray or detected in the G-Box system (Syngene, Frederick, MD). Band intensities were analyzed with Gene Tools software (Syngene). Raw volume with background subtracted was normalized to GAPDH, with vehicle-treated cells set as 100%.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!