Transfected hBMSCs were lysed with radio immunoprecipitation buffer (Beyotime Institute of Biotechnology) and a Bradford kit (Beyotime Institute of Biotechnology) was used to determine total protein concentration. Proteins (20 µg/lane) were isolated by SDS-PAGE on 10% gels and were electrotransferred onto nitrocellulose membranes (cat. no. IPVH00010; EMD Millipore); membranes were then blocked with 1% BSA (Beyotime Institute of Biotechnology) to ensure non-specific binding for 60 min at room temperature. Membranes were incubated with anti-rabbit alkaline phosphatase (ALP; 1:1,000; cat. no. ab75699; Abcam), anti-rabbit RUNX2 (1:1,000; cat. no. ab192256; Abcam) and anti-rabbit GAPDH (1:20,000; cat. no. ab181602; Abcam) primary antibodies at 4°C overnight. Anti-HRP (1:2,000; cat. no. ab181658; Abcam) secondary antibody was used to incubate the membrane for 1 h at room temperature. All antibodies were diluted with dH2O (Takara Bio, Inc.). Protein bands were observed by enhanced chemiluminescence (Beyotime Institute of Biotechnology) and exposed on X-ray film (Kodak). The relative grey values were analyzed by ImageJ 1.48 software (National Institutes of Health).
Enhanced chemiluminescence
Manufactured by Beyotime
Sourced in China, United States
Enhanced chemiluminescence is a laboratory technique used to detect and quantify specific proteins or molecules in a sample. It generates a luminescent signal in the presence of the target analyte, which can be measured and analyzed to provide information about the sample's composition. This method is commonly used in various fields, such as biochemistry, molecular biology, and immunology, for applications like Western blotting, ELISA, and protein detection.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (19 mentions)
Pvdf membrane, by Merck Group (19 mentions)
Ripa lysis buffer, by Beyotime (17 mentions)
Bca protein assay kit, by Beyotime (12 mentions)
Polyvinylidene fluoride membrane, by Merck Group (8 mentions)
Polyvinylidene difluoride membrane, by Merck Group (8 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (6 mentions)
Quantity one software, by Bio-Rad (5 mentions)
β actin, by Beyotime (5 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (5 mentions)
Ab6721, by Abcam (4 mentions)
Ab8227, by Abcam (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Lysis buffer, by Beyotime (4 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Bicinchoninic acid kit, by Beyotime (3 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (3 mentions)
P akt, by Cell Signaling Technology (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
124 protocols using enhanced chemiluminescence
1
Western Blot Analysis of hBMSCs
2
Quantification of Lung Protein Markers
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Lung tissue from mice was ground into homogenates at the condition of 4°C for protein detection [26 (link)]. Total protein was extracted via a Total Protein Extraction Kit (Invent Biotechnologies Inc., USA) according to the manufacturer's protocol. Enhanced BCA Protein Assay Kit (Beyotime, Jiangsu, China) was used for concentration detection. Protein samples were then separated by SDS-PAGE gel electrophoresis on 12% glycine-based gels and transferred to nitrocellulose membranes (Millipore Corp., Billerica, MA, USA). Membranes were blocked in 5% (w/v) skimmed milk for 2 h and incubated with antibodies in 5% (w/v) BSA overnight at 4°C. Anti-Foxp3 antibody, anti-RORγt antibody, and anti-GAPDH antibody were purchased from Abcam (United Kingdom). Membranes were then incubated with secondary HRP-labeled antibody (Sigma-Aldrich, St. Louis, MO, USA) in 5% (w/v) BSA for 1 h. Afterwards, enhanced chemiluminescence (Beyotime, Jiangsu, China) was developed on membranes. The intensity of bands was quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Quantifying Renal Protein Expression
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Kidney tissue and harvested cells were lysed on ice with radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology) and the lysates were centrifuged at 12,000 × g for 15 min at 4°C. The protein concentration was measured using a BCA assay kit (Beyotime Institute of Biotechnology). A total of 35 µg protein/lane was loaded on an 10% SDS-PAGE gel for electrophoresis and then transferred to a nitrocellulose membrane (EMD Millipore, Billerica, MA, USA). The blots were blocked in TBS-T (0.1% Tween 20) buffer with 5% skimmed milk at room temperature for 1 h and then incubated overnight with primary antibodies against e-NOS (1:500), phopho-Ser1177 eNOS (1:200) and β-actin (1:500) at 4°C. The blots were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000) for 1 h at room temperature. The immunoblots were visualized by enhanced chemiluminescence (Beyotime Institute of Biotechnology) and quantified with ImageJ software. The protein expression levels were normalized to β-actin.
4
Protein Expression Analysis in 95D Cells
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Whole cell, nuclear and cytosolic protein extracts of 95D cells were prepared using RIPA lysis buffer (Beyotime, China) containing phenylmethanesulfonyl fluoride. Equal amount of proteins were loaded onto 10% polyacrylamide gels for separation, and then transferred to the polyvinylidene fluoride membranes. These membranes were incubated with anti-WIF-1, anti-E-cadherin, anti-vimentin, anti-c-myc, anti-fibronectin, anti-β-catenin, anti-Snail (Cell Signaling Technology, USA), anti-GAPDH (Santa Cruz Biotechnology, USA) at 4°C overnight. The next day, membranes were incubated with horse reddish peroxidase-conjugated secondary antibodies (Zhongshan Golden Bridge Biotechnology, China) for 1 h at room temperature. Finally, protein bands were visualized using enhanced chemiluminescence (Beyotime, China) according to the manufacturer's protocol. GAPDH signal was used as the loading control.
5
Western Blot Quantification of Protein Levels
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The total protein quantity from HCC cells was determined by the bicinchoninic acid assay. The proteins were separated on sodium dodecyl sulfate gel and transferred onto the PVDF membranes. Proteins on membranes were blocked with 5% non-fat milk for 2 hours, probed with primary antibodies (1: 1000) overnight at 4°C and thereafter incubated with the secondary antibody (1: 10000) for 1 hour. The enhanced chemiluminescence (Beyotime) was used to visualize the brands. The antibodies used are specified in Supplementary Table 4
6
Western Blot Analysis of Apoptotic Markers
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Sodium dodecyl sulfate–polyacrylamide gel electrophoresis （SDS-PAGE） was
used to isolate the equal amounts of protein and transfer it to
polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The
membranes were blocked with 5% skim milk at room temperature, but
ser404p-tau protein membrane was blocked with 5%
bovine serum albumin (BSA). Then, incubate with primary antibody
overnight at 4°C (rabbit anti CD95, 1:300, Santa Cruz Biotechnology,
Santa Cruz, CA, USA; rabbit anti-CD95L, 1:400, rabbit anti-cleaved
caspase-3, 1:1000, rabbit anti-total tau, 1:1000, rabbit
anti-ser404p-tau, 1:1000, Abcam, Cambridge, MA, USA).
Then, the membranes were incubated with anti-rabbit secondary antibody
(1:500, Proteintech, Chicago, IL, USA) at room temperature for 1 h.
Finally, the bands were detected using enhanced chemiluminescence
(Beyotime, Shanghai, China). Image J (National Institutes of Health,
Bethesda, MD, USA) was used for quantitative band intensity;
glyceraldehyde 3-phosphate dehydrogenase (GAPDH，1:500, Abcam,
Cambridge, MA, USA) was used as the sample control.
used to isolate the equal amounts of protein and transfer it to
polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The
membranes were blocked with 5% skim milk at room temperature, but
ser404p-tau protein membrane was blocked with 5%
bovine serum albumin (BSA). Then, incubate with primary antibody
overnight at 4°C (rabbit anti CD95, 1:300, Santa Cruz Biotechnology,
Santa Cruz, CA, USA; rabbit anti-CD95L, 1:400, rabbit anti-cleaved
caspase-3, 1:1000, rabbit anti-total tau, 1:1000, rabbit
anti-ser404p-tau, 1:1000, Abcam, Cambridge, MA, USA).
Then, the membranes were incubated with anti-rabbit secondary antibody
(1:500, Proteintech, Chicago, IL, USA) at room temperature for 1 h.
Finally, the bands were detected using enhanced chemiluminescence
(Beyotime, Shanghai, China). Image J (National Institutes of Health,
Bethesda, MD, USA) was used for quantitative band intensity;
glyceraldehyde 3-phosphate dehydrogenase (GAPDH，1:500, Abcam,
Cambridge, MA, USA) was used as the sample control.
7
Western Blotting Analysis of EMT Markers
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Western blotting was performed as previously described (8 (link)). In brief, protein lysates (10–30 µg) were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were subsequently transferred onto polyvinylidene difluoride membranes (EMD Millipore). The membranes were first incubated overnight with primary antibodies specific for E-cadherin (1:10,000; cat. no. 20874-1-AP), vimentin (1:5,000; cat. no. 10366-1-AP), BTG1 (1:1,000; cat. no. 14879-1-AP), CD63 (1:1,000; cat. no. 25682-1-AP), TSG101 (1:3,000; cat. no. 14497-1-AP) and GAPDH (1:6,000; cat. no. 60004-1-Ig) that had been diluted in a 5% low-fat milk-TBS with 0.1% Tween-20 solution and then with a horseradish peroxidase-conjugated secondary antibody (1:3,000; cat. no. PR30009). The labeled protein bands were visualized using enhanced chemiluminescence (Beyotime Institute of Biotechnology), and band intensities were semi-quantified using ImageJ software (version 1.49p; National Institutes of Health). All primary and secondary antibodies were purchased from Wuhan Sanying Biotechnology.
8
Western Blot Analysis of STAT3 and NF-κB Signaling
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PBMCs were lysed on ice for 30 min using RIPA lysis buffer (Beyotime) with protease inhibitors (Beyotime) and phosphatase inhibitors cocktail (Beyotime), and the protein concentration was determined by a bicinchoninic acid assay kit (Biosharp). The proteins (50 µg per lane) were mixed with one-fifth volume of 5× loading buffer, separated by 10% SDS-PAGE, and were then transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked for 1 h with 5% nonfat milk at room temperature and were incubated overnight at 4°C with primary antibodies against STAT3 rabbit mAb (79D7, Cell Signaling Technology), phospho-STAT3-Tyr705 rabbit mAb (D3A7, Cell Signaling Technology), NF-κB p65 rabbit mAb (D14E12, Cell Signaling Technology), anti-NF-kB p65-phospho-S536 antibody (EP2294Y, Abcam), and β-actin rabbit monoclonal antibody (Beyotime). After 3 washes with Tris-buffered saline with 0.1% Tween® 20 detergent, the membranes were incubated for 1 h at room temperature with anti-rabbit horseradish peroxidase-conjugated secondary antibody (GE) and were detected with enhanced chemiluminescence (Beyotime). The results were analyzed using Image Studio software.
9
Western Blot Analysis of EZH2 Expression
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Proteins were extracted from cells using Radio‐Immunoprecipitation Assay buffer (RIPA, Beyotime Institute of Biotechnology), and the concentration of proteins was calculated by a BCA protein assay kit (Beyotime Institute of Biotechnology). An equal amount of proteins (20 μg/lane) was subjected to 12% SDS‐PAGE, followed by an electrophoretic transfer onto a polyvinylidene fluoride membrane. After being blocked with 5% non‐fat milk dissolved in Tris‐buffered saline containing 0.1% Tween‐20 (TBST) for 1 hour at room temperature, membranes were incubated with rabbit polyclonal antibodies against EZH2 (ab186006, 1:1,000, Abcam) or β‐actin (ab8227, 1:2,000, Abcam) overnight at 4°C. The next morning, membranes were washed with TBST three times and probed with horseradish peroxidase‐conjugated (HRP) goat anti‐rabbit IgG H & L secondary antibody (ab6721, 1:10,000, Abcam) for 1 hour at 37°C. After being washed with TBST, immunoreactive bands were developed by enhanced chemiluminescence (Beyotime Institute of Biotechnology) and the grey values of each blot were calculated by ImageJ version 1.50 (National Institutes of Health). The relative expression of the target protein was calculated by normalization to β‐actin.
10
Protein Extraction and Western Blot Analysis
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Total protein was extracted in RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) on ice for 30 min and then centrifuged at 12,000 × g for 10 min at 4°C. Total protein was quantified using a BCA assay (Beyotime Institute of Biotechnology) and total protein samples (50 µg) were subjected to 8–12% SDS-polyacrylamide gel electrophoresis and then transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk in TBST, and incubated at 4°C overnight with the following primary antibodies: MDM4 (cat. no. sc-14738; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), B-cell lymphoma 2-associated X protein (Bax; cat. no. sc-6236, 1:1,000), p53 (cat. no. sc-47698; 1:1,000) and GAPDH (cat. no. sc-51631; 1:5,000; all Santa Cruz Biotechnology). The membranes were washed with TBST for 15 min and then incubated with the anti-rabbit horseradish peroxidase secondary antibody (cat. no. sc-2004; 1:5,000; Santa Cruz Biotechnology, Inc.) for 1 h at 37°C. The protein bands were detected with enhanced chemiluminescence (Beyotime Institute of Biotechnology).
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