According to the results of proteome sequencing, WB was performed on the selected eight protein targets. Tissues were collected and homogenized using RIPA buffer (Beyotime Institute of Biotechnology). Equal amounts of total protein (30 µg/lane) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, USA). The primary antibodies were anti-IGFBP2 (ab227881, 1:1,000, Abcam, USA), anti-TPM3 (ab190993, 1:1,000, Abcam, USA), anti-ALDOB (ab129728, 1:1,000, Abcam, USA), anti-FAH (PA5-42049, 1:1,000, Invitrogen, USA), anti-SOD3 (14316-1-AP, 1:1,000, Proteintech, China), anti-SPARC (ab55847, 1:1,000, Abcam, USA), anti-APOE (ab52607, 1:1,000, Abcam, USA), anti-RBP4 (ab154914, 1:1,000, Abcam, USA), and anti-GAPDH (ab8245, 1:5,000, Abcam, USA). And the secondary antibody goat was anti-mouse IgG (PAB31797; Abnova, China). The bands were then developed using enhanced chemiluminescence chromogenic substrate (GE Healthcare, UK) and analyzed by the Image J software. GAPDH was used as control in this study.
Enhanced chemiluminescence chromogenic substrate
Manufactured by GE Healthcare
Sourced in United Kingdom
Enhanced chemiluminescence chromogenic substrate is a laboratory reagent used to detect and visualize proteins in Western blot analysis. It functions by producing a colorimetric or luminescent signal upon exposure to the target protein, enabling its detection and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Sds page, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab129728, by Abcam (1 mentions)
Ab55847, by Abcam (1 mentions)
Ab52607, by Abcam (1 mentions)
Pa5 42049, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab8245, by Abcam (1 mentions)
Blocking reagent, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Bca assay kit, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by ABclonal (1 mentions)
Gapdh, by ABclonal (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Bio-Rad (1 mentions)
Bicinchoninic acid assay kit, by Thermo Fisher Scientific (1 mentions)
P ampk, by Abcam (1 mentions)
6 protocols using enhanced chemiluminescence chromogenic substrate
1
Protein Expression Analysis by Western Blot
2
Quantitative Protein Analysis of Tumor Samples
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Total proteins were isolated from tumor tissues or cells using cell lysis buffer containing 1% protease inhibitor (Thermo Fisher, Wilmington, DE, USA) and quantified using BCA assay kit (Sigma) according to the instructions. Denatured proteins were separated by SDS-PAGE gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with blocking reagent (Thermo Fisher) for 1 h at room temperature and then incubated with primary antibodies against PTEN or β-actin (Cell Signaling Technology, Danvers, MA, USA) overnight at 4℃, followed by interacting with horseradish peroxidase conjugated secondary antibodies (Cell Signaling Technology) for 2 h at room temperature. The protein blots were detected using enhanced chemiluminescence chromogenic substrate (GE Healthcare, Amersham, UK) and investigated by Image Lab software (Bio-Rad).
3
Western Blot Protein Expression Analysis
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The protein expression of cells or tissues was detected by western blot analysis
as previously described.19 (link)
In brief, cells or tissue samples were collected and homogenized using
radioimmunoprecipitation assay buffer. Equal total proteins (30 µg/lane) were
separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad,
USA) and transferred onto the polyvinylidene difluoride membrane (Millpore,
USA). Next, the membranes were blocked by 5% bovine serum albumin (BSA) for 2
hours at room temperature, followed by incubation with primary antibodies
against Wave2 (1:500, ABclonal, China) and GAPDH (1:1000, ABclonal, China) at 4
°C overnight. After washing with 1× phosphate-buffered saline (PBS) for 3 times,
the membranes were incubated with HRP-conjugated secondary antibodies (1:5000,
ABclonal, China) for 2 hours at room temperature. The bands were then developed
using enhanced chemiluminescence chromogenic substrate (GE Healthcare, UK) and
analyzed by the Image J software. GAPDH was used as a control in this study.
as previously described.19 (link)
In brief, cells or tissue samples were collected and homogenized using
radioimmunoprecipitation assay buffer. Equal total proteins (30 µg/lane) were
separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad,
USA) and transferred onto the polyvinylidene difluoride membrane (Millpore,
USA). Next, the membranes were blocked by 5% bovine serum albumin (BSA) for 2
hours at room temperature, followed by incubation with primary antibodies
against Wave2 (1:500, ABclonal, China) and GAPDH (1:1000, ABclonal, China) at 4
°C overnight. After washing with 1× phosphate-buffered saline (PBS) for 3 times,
the membranes were incubated with HRP-conjugated secondary antibodies (1:5000,
ABclonal, China) for 2 hours at room temperature. The bands were then developed
using enhanced chemiluminescence chromogenic substrate (GE Healthcare, UK) and
analyzed by the Image J software. GAPDH was used as a control in this study.
4
Western Blot Analysis of Cellular Proteins
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Cell proteins were prepared by RIPA lysis buffer with 1% phenylmethylsulfonyl fluoride and quantified using bicinchoninic acid assay kits (Thermo Fisher) following given instructions. Proteins were denatured at 95℃ for 10 min in protein loading buffer (Invitrogen). Denatured samples were then loaded onto a 10% SDS-PAGE gel and then transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4℃ with primary monoclonal antibodies against NS3, NS5A, IRF5, double-stranded RNA-dependent protein kinase (PKR), 2′–5′-oligoadenylate synthetases (OAS), myxovirus protein A (MxA), or β-actin (Cell Signaling Technology, Danvers, MA, USA). After three washes in Tris-buffer saline containing 0.1% Tween 20 (TBST), membranes were hatched with secondary antibodies conjugated by horseradish peroxidase for 2 h at room temperature, and then washed three times in TBST before incubation with enhanced chemiluminescence chromogenic substrate (GE Healthcare, Amersham, UK) for visualization of immunoreactivity and densitometry analysis using Image Lab software (Bio-Rad, Hercules, CA, USA).
5
Western Blot Analysis of Protein Expression
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The protein expression of cells or tissues was detected by using western blot as previously described (Jing W. et al., 2019 (link)). Cells or tissue samples were collected and homogenized using RIPA buffer. Equal total proteins (30 μg/lane) were separated by SDS-PAGE (Bio-Rad, United States), and transferred onto the PVDF membrane. Next, the membranes were blocked by 5% BSA for 2 h at room temperature, followed by incubation with primary antibodies against ER-α (1:1000, Abcam, United States), LKB1 (1:1000, Abcam, United States), p-LKB1 (1:1000, Abcam, United States), AMPK (1:1000, Abcam, United States), p-AMPK (1:1000, Abcam, United States), mTOR (1:1000, Abcam, United States), p-mTOR (1:1000, Abcam, United States), HAND2 (1:1000, Abcam, United States), Lamin B1 (1:5000, Abcam, United States), and GAPDH (1:5000, Abcam, United States) at 4°C overnight. After washing with 1 × PBS for three times, the membranes were incubated with HRP-conjugated secondary antibodies (1:5000, Abcam, United States) for 2 h at room temperature. The bands were then developed using enhanced chemiluminescence chromogenic substrate (GE Healthcare, United Kingdom) and analyzed by the Image J software. Lamin B1 or GAPDH was used as a control in this study.
6
Western Blot Analysis of Spinal Cord Proteins
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Total proteins from the spinal cord tissues or cells were extracted using RIPA Lysis Buffer (ThermoFisher) and were quantified by DC protein assay. Western blot was carried out as previous described[34 (link)]. The primary antibodies were listed in Table 2 and the bands were then developed using enhanced chemiluminescence chromogenic substrate (GE Healthcare, UK) and analyzed by the Image J software.
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