Total protein extracts were obtained treating each tissue sample with total lysis buffer (Pierce Ripa buffer, Thermo Scientific, Rockford, IL, USA) supplemented with protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL, USA). After homogenization and centrifugation at 14,000 rpm for 15 min at 4 °C, the protein concentration was measured by a standard Bradford assay (Bio-Rad, Milan, Italy). Aliquots of 50 µg of total protein extracts from each sample were denaturated in 5× Laemmli sample buffer and loaded into 4–12% pre-cast polyacrylamide gels (Bio-Rad, Milan, Italy) for western blot analysis. Cannabinoid receptor I (Abcam, Cambridge, UK), cleaved caspase-3 (Asp175), Bax, ERK1/2, p-ERK1/2 (Thr202/Tyr204), p38α MAPK, p-p38 (Thr180/Tyr182) MAPK, Akt, p-Akt (Thr308), p-Akt (Ser473), β-actin (Cell Signaling Technology, Beverly, MA, USA) and Bcl2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), were used as primary antibodies. After overnight incubation, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Milan, Italy). The proteins were detected by chemiluminescence (ECL, Thermo Scientific, Rockford, IL, USA) and each protein-related signal was obtained using the Molecular Imager ChemidocTM (Bio-Rad, Milan, Italy) and normalized against β-actin protein expression.
Molecular imager chemidoctm
Manufactured by Bio-Rad
Sourced in Italy
The Molecular Imager ChemidocTM is a versatile imaging system designed for the detection and analysis of protein and nucleic acid samples in a variety of applications. It utilizes advanced imaging technology to capture high-quality images of chemiluminescent, colorimetric, and fluorescent signals.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Bio-Rad (6 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (4 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (3 mentions)
Pre cast polyacrylamide gels, by Bio-Rad (3 mentions)
Pierce ripa buffer, by Thermo Fisher Scientific (3 mentions)
Precast polyacrylamide gel, by Bio-Rad (3 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Pparγ, by Abcam (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Anti vdac1, by Abcam (1 mentions)
Anti tlr4, by Abcam (1 mentions)
Anti beclin1, by Abcam (1 mentions)
Anti pparγ, by Abcam (1 mentions)
8 protocols using molecular imager chemidoctm
1
Western Blot Analysis of Protein Signaling
2
Protein Expression Analysis in Rat Jejunal Tissue
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Protein extracts were obtained treating each jejunal tissue sample from control and treated rats with total lysis buffer (Pierce Ripa buffer, Thermo Scientific, Rockford, IL, USA) supplemented with protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL, USA).
After homogenization and centrifugation at 14,000 rpm for 15 min at 4 °C, protein concentration was measured by a standard Bradford assay (Bio-Rad, Milan, Italy). Aliquots of 50 µg of total protein extracts from each sample were denaturated in 5× Laemmli sample buffer and loaded into 4–12% pre-cast polyacrylamide gels (Bio-Rad, Milan, Italy) for western blot analysis. ODC (N-15), ZO-1 (C-19), Occludin (H-279), Claudin-1 (C-18), β-catenin (H-102), p-β-catenin (Ser 33), E-cadherin (H-108) and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as primary antibodies. After overnight incubation, the membranes were further incubated with a horseradish peroxidase-conjugated goat secondary antibody (Bio-Rad, Milan, Italy). The proteins were detected by chemiluminescence (ECL, Thermo Scientific, Rockford, IL, USA) and the densitometric analysis of each protein-related signal was obtained using the Molecular Imager ChemidocTM (Bio-Rad, Milan, Italy) and normalized against β-actin expression.
After homogenization and centrifugation at 14,000 rpm for 15 min at 4 °C, protein concentration was measured by a standard Bradford assay (Bio-Rad, Milan, Italy). Aliquots of 50 µg of total protein extracts from each sample were denaturated in 5× Laemmli sample buffer and loaded into 4–12% pre-cast polyacrylamide gels (Bio-Rad, Milan, Italy) for western blot analysis. ODC (N-15), ZO-1 (C-19), Occludin (H-279), Claudin-1 (C-18), β-catenin (H-102), p-β-catenin (Ser 33), E-cadherin (H-108) and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as primary antibodies. After overnight incubation, the membranes were further incubated with a horseradish peroxidase-conjugated goat secondary antibody (Bio-Rad, Milan, Italy). The proteins were detected by chemiluminescence (ECL, Thermo Scientific, Rockford, IL, USA) and the densitometric analysis of each protein-related signal was obtained using the Molecular Imager ChemidocTM (Bio-Rad, Milan, Italy) and normalized against β-actin expression.
3
Protein Expression Analysis in Tissues
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Total protein extracts were obtained treating each tissue sample with total lysis buffer (Pierce Ripa buffer, Thermo Scientific, Rockford, IL, USA) supplemented with protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL, USA). After homogenization and centrifugation, protein concentration was measured by a standard Bradford assay (Bio-Rad, Milan, Italy). Aliquots of 50 µg of total protein extracts from each sample were denaturated in Laemmli sample buffer and loaded into 4–12% pre-cast polyacrylamide gels (Bio-Rad, Milan, Italy) for western blot analysis. CB2-R (cat. n° ab 3561, Abcam, Cambridge, MA), PPAR-γ (cat. n° #2443, Cell Signaling Technology, Beverly, MA, USA), and β-actin (cat. n° #4970, Cell Signaling Technology, Beverly, MA, USA) were used as primary antibodies. After overnight incubation, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Milan, Italy). The proteins were detected by chemiluminescence (ECL, Thermo Scientific, Rockford, IL, USA) and each protein-related signal was obtained using the Molecular Imager ChemidocTM (Bio-Rad, Milan, Italy) and normalized against β-actin protein expression.
4
Protein Expression Analysis of Adipose Tissue
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Protein lysates were prepared by homogenizing adipose tissue in lysis buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5μg/ml of aprotinin, 5μg/ml of leupeptin). Tissue and cell debris was removed by centrifugation. Protein concentrations were estimated by the Bio-Rad protein assay using BSA as standard. The lysates were boiled for 5 min in 1×SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% SDS, 0.01% bromophenol blue) containing 5% Beta-mercaptoethanol.
Equal amount of protein (50 μg) were resolved on SDS–PAGE and transferred to polyvinylidenefluoride (PVDF) filters. After transfer, the membranes were blocked for 2 hours in 5% non-fat dry milk in Tris-buffered saline (TBS) and then incubated in primary antibodies overnight at 4°C.The primary antibodies were diluted 1 : 500 in blocking buffer and were directed against the following proteins: CB2-R, NOS1 and GAPDH. After overnight incubation, the membranes were further incubated with a horseradish peroxidase conjugated secondary antibody. The proteins were detected by chemiluminescence (ECL) and the densitometric analysis of each protein-related signal was obtained using the Molecular Imager Chemidoc TM (Bio-Rad Laboratories) and normalized against GAPDH expression.
Equal amount of protein (50 μg) were resolved on SDS–PAGE and transferred to polyvinylidenefluoride (PVDF) filters. After transfer, the membranes were blocked for 2 hours in 5% non-fat dry milk in Tris-buffered saline (TBS) and then incubated in primary antibodies overnight at 4°C.The primary antibodies were diluted 1 : 500 in blocking buffer and were directed against the following proteins: CB2-R, NOS1 and GAPDH. After overnight incubation, the membranes were further incubated with a horseradish peroxidase conjugated secondary antibody. The proteins were detected by chemiluminescence (ECL) and the densitometric analysis of each protein-related signal was obtained using the Molecular Imager Chemidoc TM (Bio-Rad Laboratories) and normalized against GAPDH expression.
5
Protein Expression Profile in IBS Rats
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Protein extracts were obtained from colon tissue samples of control, IBS-Std, and IBS-KD rats using standard procedure [7 (link)]. Aliquots of 50 µg of total protein extracts from each sample were loaded into 4–15% pre-cast polyacrylamide gels (Bio-Rad, Milan, Italy) for western blot analysis. Anti-PrxIII (Ab FRONTIER, Seoul, Korea), anti-PGC-1α (Ab NOVUS, Centennial, CO, USA), anti-COX-2, anti-TLR-4, anti-PPAR-γ, anti-COX-4, anti-BECLin-1, anti-LC3, anti-VDAC1 (Abcam, Cambridge, UK), and anti-β-actin (Cell Signaling, Danvers, MA, USA) were used as primary antibodies. The proteins were detected by chemiluminescence (ECL, Thermo Scientific, Rockford, IL, USA), and the densitometric analysis of each protein-related signal was obtained using the Molecular Imager ChemidocTM (Bio-Rad, Milan, Italy) and normalized against β-actin expression.
6
Western Blot Analysis of Protein Expression
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Protein samples were subjected to electrophoresis using SDS-PAGE gel and subsequently transferred onto a PVDF (polyvinylidene difluoride) membrane (Bio-Rad Laboratories, Milan, Italy) and probed with anti-β-catenin, anti-c-myc and anti-GAPDH primary antibodies (Cell Signaling, Beverly, MA, USA). After overnight incubation, a horseradish peroxidase-conjugated secondary antibody (Bio-Rad Laboratories) was used. After chemiluminescence and densitometric analysis, the signal of each protein was obtained using the Molecular Imager ChemidocTM (Bio-Rad Laboratories) and normalized against GAPDH expression.
7
Protein Expression Analysis in Rat Liver
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Protein extracts were obtained from liver samples of Ctrl, Fructose, HF, HF-F, and HF-F-D rats using standard procedure [16 (link)]. Aliquots of 50 μg of total protein from each sample were loaded into 4–15% pre-cast polyacrylamide gels (Bio-Rad, Milan, Italy) for Western blot analysis. Anti-PrxIII (LF-PA0030,Ab FRONTIER, Seoul, Korea), anti-PGC-1α (NBBP1-04676, Ab NOVUS, Centennial, CO, USA), anti-TFAM (74955), anti-COX-IV (48445), anti-Beclin-1 (37385), anti-LC3 (127414), and anti-β-actin (4970) (Cell Signaling, Danvers, MA, USA) were used as primary antibodies. The protein signals were detected by chemiluminescence (Clarity Western ECL substrate, Bio-Rad, Milan, Italy); the densitometric analysis was obtained using the Molecular Imager ChemidocTM (Bio-Rad, Milan, Italy), and data were normalized against β-actin expression.
8
Serotonin Signaling Regulation in Colon and Brain
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Protein extracts were obtained using a standard procedure from colon and brain samples of Ctrl-Std, KD, IBS-Std, and IBS-KD rats. For Western blot analysis, the aliquots of 50 µg of total protein extracts from each sample were loaded into 10% pre-cast polyacrylamide gels (Bio-Rad, Milan, Italy). Anti-5-HT3B, anti-5-HT4, anti-SERT (Abcam, Cambridge, UK), anti-BDNF (MyBioSource, San Diego, CA, USA), anti-TrkB (Thermo Scientific, Rockford, IL, USA), anti-β-actin, and anti-GAPDH (Cell Signaling, Danvers, MA, USA) were used as primary antibodies. The proteins were detected by chemiluminescence (ECL, Bio-Rad, Milan, Italy), and the densitometric analysis of each protein-related signal was obtained using the Molecular Imager ChemidocTM (Bio-Rad, Milan, Italy) and normalized against β-actin or GAPDH expression.
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