Total protein was extracted using a radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China) with addition of PMSF and protease inhibitor; the protein concentration was determined using a BCA assay kit (Beyotime, Shanghai, China). The proteins were separated on a 7.5–10% SDS polyacrylamide gel electrophoresis, transferred on PVDF membranes at 200 mA at 4 °C and closed in fast closing solution (New Cell Molecular Biotechnology Co., Ltd., Shanghai, China) for 30 min. Protein detection was performed using enhanced chemiluminescence detection reagents (Beyotime, Shanghai, China). Polyclonal rabbit anti-GAPDH antibody was used as a loading control. Western blots were developed and quantified with BioSpectrum 810 Imaging System using VisionWorksLS 7.1 software (UVP LLC, Upland, CA, USA). The standard markers for protein molecular masses were supplied by Thermo (Cat# 26617, Thermo Fisher, Carlsbad, CA, USA). The membranes were probed with the required antibodies: OXPHOS antibody cocktail (Cat# ab110413, abcam, Cambridge, UK), GAPDH (Cat# ab9485, abcam, Cambridge, UK). The horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and goat anti-mouse IgG antibody were supplied by Beyotime (Beyotime, Shanghai, China).
Enhanced chemiluminescence detection reagent
Manufactured by Beyotime
Sourced in China
Enhanced chemiluminescence detection reagents are a set of reagents used for the detection and visualization of proteins or other biomolecules in Western blot analysis. These reagents generate a luminescent signal upon interaction with the target analyte, allowing for sensitive and quantitative detection.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Bca kit, by Beyotime (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Anti bax, by Cell Signaling Technology (2 mentions)
Anti bcl 2, by Cell Signaling Technology (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Oxphos antibody cocktail, by Abcam (1 mentions)
Goat anti mouse igg antibody, by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Horseradish peroxidase, by Beyotime (1 mentions)
Anti sfrp1, by Abcam (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Mouse monoclonal antibody against β actin, by Merck Group (1 mentions)
Image lab 3, by Bio-Rad (1 mentions)
P0012, by Beyotime (1 mentions)
22 protocols using enhanced chemiluminescence detection reagent
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of SFRP1
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Proteins were isolated and resolved by SDS–PAGE, and then transferred to PVDF membranes. The PVDF membrane was blocked for 1 h in 5% non-fat milk dissolved in tris-buffered saline-Tween 20. The PVDF membrane was incubated with anti-SFRP1 (Abcam, Cambridge, MA, USA) or anti-GAPDH antibodies (Abcam) at 4°C overnight. After washing, the PVDF membrane was incubated with a secondary HRP-conjugated antibody for 1 h at 25°C. Immunoreactive bands were visualized with enhanced chemiluminescence detection reagents (Beyotime, Shanghai, China). The experiment was carried out at least three times.
3
Western Blot Analysis of LC3B in L-02 Cells
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L-02 cells in each 25-cm2 culture flask were collected and proteins were extracted with 1 ml mixture of RIPA lysis buffer (P0013B; Beyotime Institute of Biotechnology, Haimen, China) and phenylmethylsulfonyl fluoride (final concentration, 1 mmol/l). Protein concentration was determined using a bicinchoninic acid protein assay kit (P0012; Beyotime Institute of Biotechnology). Thirty micrograms of protein samples were separated by SDS-PAGE and then electro-transferred onto the polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA, USA). Membranes were blocked for 1 h with 5% skimmed milk in TBST buffer [50 mmol/l Tris (pH 7.6), 150 mmol/l NaCl and 0.1% Tween-20] and incubated with rabbit monoclonal antibodies against human LC3B (Cell Signaling Technology, Inc., Beverly, MA, USA) overnight at 4°C. β-actin was used as an internal control and was detected using a mouse monoclonal antibody against β-actin (Sigma, St. Louis, MO, USA). Following incubation with secondary antibodies, enhanced chemiluminescence detection reagents (Beyotime Institute of Biotechnology) were used to detect the signals. The intensity of the signals was quantified with Image Lab 3.0 software (Bio-Rad Laboratories, Inc.).
4
Western Blot Analysis of Cell Signaling
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The HUVECs in each group were lysed using RIPA lysis buffer (Solarbio Biotechnology, Beijing, China). Equivalent amounts of proteins were separated with 12% SDS-polyacrylamide gels and then transblotted onto a PVDF membrane. The membranes were incubated with 5% dried nonfat milk buffer for 1 h to prevent nonspecific binding. They were then incubated with the relevant primary antibodies and with a secondary antibody. The protein bands in the membranes were detected by enhanced chemiluminescence detection reagents (Beyotime, Shanghai, China) and analyzed with Image J. Anti-Bcl-2, anti-Bax and anti-cleaved caspase-3 were from Cell Signaling Technology (Beverly, MA, USA). Anti-VCAM and anti-ICAM were from Abcam (Cambridge, MA, USA). Anti-p-IKBα, anti-IKBα, and anti-IL-8 were from Bioworld Technology (St. Louis Park, MN, USA).
5
Cytochrome C Quantification by Western Blot
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The protein concentration was determined using a BCA Protein Assay kit (Lanbao Biotechnology, Shanghai, China) according to according to the manufacturer’s protocol. The protein was electrophoresed using 15% SDS-PAGE (as described above) and transferred onto nitrocellulose membranes (Beyotime Institute of Biotechnology, Shanghai, China). Following blocking, the membranes were incubated for 4 h at 37°C with the primary antibody, which was goat anti-mouse monoclonal anti-cytochrome C (1:400; IM 94638; Beyotime Institute of Biotechnology), and the secondary antibody (1:5,000; 572909; Beyotime Institute of Biotechnology), which was goat anti-rat alkaline phosphatase-conjugated immunoglobulin E. The blots were visualized using enhanced chemiluminescence detection reagents (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. The SynGene GeneTools analysis software-version 3.02.00 (SynGene, Frederick, MD, USA) was used to analyze the images and perform calculations.
6
Western Blot Analysis of pBMSCs
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At the end of incubation, the pBMSCs were harvested and washed twice with PBS. Then Western blot was conducted as previously described [26 (link)]. Cells were lysed by lysis buffer, and the cell lysates were centrifuged to remove insoluble materials and the protein concentration of each sample was measured. Equal protein amounts of each sample were separated by SDS-PAGE and electroblotted to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk in TBST for 2 h at room temperature and then incubated with different primary antibodies, including anti-CaSR (1 : 500), anti-cyclin D1 (1 : 2000), anti-p21 (1 : 2000), anti-ERK1/2 (1 : 2000), and anti-phospho-ERK1/2 (1 : 2000), at 4°C overnight. Then the membranes were washed and incubated with different HRP-labeled secondary antibodies at room temperature for 1 h. Finally, the proteins were detected using the enhanced chemiluminescence detection reagents (Beyotime Institute of Biotechnology, Jiangsu, China) with a FluorChem M Fluorescent Imaging System (ProteinSimple, Santa Clara, CA, USA). Protein expressions were analyzed using ImageJ software.
7
Western Blot Analysis of Protein Expression
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Radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology) was used to collect the protein from VSMCs. Then the protein concentration was measured by bicinchoninic acid (BCA) kit (Beyotime Biotechnology). The proteins (30 μg/lane) were separated by 10% sodium dodecyl sulfate polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane (EMd Millipore). After blocked in skimmed milk at room temperature for 2 h, the membranes were incubated with primary antibodies against Wnt5a (1: 1, 000; cat. no. ab179824, Abcam), EIF4A3 (1: 1, 000; cat. no. ab32485, Abcam), FBL (1: 500, cat. no. ab4566, Abcam), NOP 56 (1: 1, 000; cat. no. A18693, ABclonal), FUS (1: 1, 000; cat. no. ab124923, Abcam), IGF2BP3 (1: 1, 000; cat. no. A4444, ABclonal), LIN28 (1: 1, 000; cat. no. ab63740, Abcam), LIN28B (1: 2, 000; cat. no. ab191881, Abcam) and GAPDH (1: 1, 000; cat. no. cat. no. ab8245, Abcam) overnight at 4 °C. On the following day, the membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:1,000; cat. no. ab6721; Abcam). The protein bands were visualized by enhanced chemiluminescence detection reagents (Beyotime Biotechnology) and analyzed with Image J software (National Institutes of Health). GAPDH served as the internal reference.
8
Protein Expression Analysis by Western Blot
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Total cellular proteins of each sample were separated by SDS-PAGE electrophoresis, and western blots were conducted as our previous study described. After blocking, the membranes were overlaid with primary antibodies for cleaved caspase-3 (1:1000, Hua An, Hangzhou, China), STAT3 (1:1000, Hua An, Hangzhou, China), and p53 (1:2000, Hua An, Hangzhou, China) overnight at 4°C, then incubated with secondary antibody at 37°C for 1 h. Proteins were visualized by enhanced chemiluminescence detection reagents (Beyotime, China). The endogenous control used was β-actin.
9
Western Blot Analysis of Signaling Proteins
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Total cellular proteins of each sample were separated by SDS-PAGE electrophoresis, and western blots were conducted as our previous study described [22 (link)]. The proteins were transferred onto polyvinylidene fluoride membranes. After blocking, the membranes were overlaid with primary antibodies for phosphorylated or total p38, JNK, and ERK (1 : 500) overnight at 4°C, then incubated with secondary antibody at 37°C for 1 h. Proteins were visualized by enhanced chemiluminescence detection reagents (Beyotime). β-Actin was used as the endogenous control.
10
Analyzing MMP16 and MMP2 Protein Levels
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Forty-eight hours after miRNA mimics or miRNA inhibitor transfection, total proteins were isolated from tissues and cell lines with radioimmunoprecipitation (RIPA) lysis buffer (P0013B, Beyotime, People’s Republic of China). The supernatants containing the whole protein extracts were obtained after centrifugation of the lysates at 12,000× g for 20 minutes at 4°C. The protein concentrations were determined by enhanced bicinchoninic acid protein assay kit (Beyotime). Heat-denatured protein samples (50 µg per lane) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) gel and transferred to an Immobilon-P transfer membrane (EMD Millipore). The membrane was blocked in 5% non-fat dry milk in Tris-buffered saline (pH 7.4) containing 0.05% Tween-20 to block nonspecific binding, followed by incubation overnight at 4°C with a primary rabbit polyclonal antibody against human MMP16 (1:200, Boster, People’s Republic of China), MMP2 (1:250, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and was blotted with goat anti-rabbit immunoglobulin G (1:3,000, Santa Cruz Biotechnology Inc., USA). Then GAPDH was used as a loading control. Signals were detected by secondary antibodies labeled with HRP, and the bound antibody was detected with the use of enhanced chemiluminescence detection reagents (Beyotime) according to the manufacturer’s instructions.
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