Total proteins were extracted using a RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China). A bicinchoninic protein assay kit was used to quantify protein concentrations. Then, the protein samples were separated by 12% SDS-PAGE and transferred to polyvinylodene difluoride membranes. The membranes were blocked with 5% skim milk powder overnight at 4°C. Primary antibodies against β-catenin (~90 kDa) and β-actin were purchased from Santa Cruz Biotechnology, Inc., (anti-β-catenin; cat. no. sc-7963; 1:200; anti-β-actin, cat. no. sc-130301; 1:10,000) and incubated overnight at 4°C. The secondary antibody, goat anti-mouse immunoglobulin (Ig) G-horseradish peroxidase, was purchased from Santa Cruz Biotechnology, Inc., (cat. no. sc-2005; 1:5,000) and incubated at 37°C for 1 h. Finally, the bands were visualized using an enhanced chemiluminescence kit (Solarbio, Beijing, China) and analyzed by Image pro plus (version 9.3; Media Cybernetics, Inc., Rockville, MD, USA).
Enhanced chemiluminescence kit
Manufactured by Solarbio
Sourced in China
The Enhanced Chemiluminescence Kit is a laboratory equipment used to detect and quantify proteins in Western blot analysis. It utilizes a chemiluminescent substrate to produce a detectable light signal upon reaction with the target proteins, allowing for sensitive and accurate protein visualization.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Horseradish peroxidase, by Proteintech (1 mentions)
Igf 1, by Abcam (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay kit, by Dingguo (1 mentions)
Ab243140, by Abcam (1 mentions)
Ab131476, by Abcam (1 mentions)
Ab6276, by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
Gapdh monoclonal antibody, by Immunoway (1 mentions)
Protein extraction kit, by Keygen Biotech (1 mentions)
Bca protein detection kit, by Keygen Biotech (1 mentions)
Image analysis system, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab205718, by Abcam (1 mentions)
17 protocols using enhanced chemiluminescence kit
1
Western Blot Analysis of β-Catenin
2
Western Blot Analysis of Srebp-1, Lamin B1, and β-Actin
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As previously described (Sun et al., 2020 (link)), sterol regulatory element-binding protein-1 (Srebp-1) and Lamin B1 or β-actin protein in both the nucleus and cytoplasm were extracted with kits, and the protein samples were separated by 10% SDS–polyacrylamide gel electrophoresis. Next, the protein samples were transferred to a PVDF membrane (0.22 µm), which was blocked with 5% skim milk for 4 h at room temperature. Subsequently, the membrane was incubated with primary antibodies, including Srebp-1 (1:1,000), β-actin (1:2,000), and Lamin B1 (1:2,000), which were diluted in Tris-buffered saline with Tween-20 containing 5% skim milk at 4°C overnight. The immune complexes were recognized by a secondary antibody (1:2,000) conjugated to horseradish peroxidase (ProteinTech, Chicago, United States). Peroxidase activity was visualized using an enhanced chemiluminescence kit (Solarbio, Beijing, China). Densitometric analysis of the immunoblots was performed using ImageJ software.
3
Molecular Profiling of Hemorrhagic Stroke
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At 72 h after ICH, the rats were killed by injection with an overdose of chloral hydrate. The brain hemispheres were harvested immediately, and total protein of the hemorrhagic hemisphere was isolated from ipsilateral brain tissues with cold RIPA buffer. A BCA protein assay kit (Dingguo, China) was used to determine protein concentration. The supernatant was subjected to SDS-polyacrylamide gel electrophoresis, and the separated proteins were transferred to a polyvinylidene difluoride filter membrane. The membranes were blocked with 5% non-fat dry milk for 1 h. Next, membranes were probed overnight with primary antibodies for zonula occluden (ZO)-1, claudin-5, brain-derived neurotrophic factor (BDNF), neurotrophic growth factor (NGF), vascular endothelial growth factor (VEGF), and insulin growth factor 1 (IGF-1) (Abcam, USA). The blots were incubated with secondary antibodies for 1 h after washing with Tris-buffered saline. An enhanced chemiluminescence kit (Solarbio, China) was used to visualize bands in immunoblots, and GAPDH (1:1,000, Santa Cruz, CA, USA) was used as the loading control.
4
Western Blot Analysis of Cell Lines
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HepG2, A549, Bel-7402, SK-Hep-1 and SMMC-7721 cells were lysed with RIPA lysis buffer (150 mM NaCl; 50 M Tris-HCl; pH, 7.5; 1% Triton X-100; 0.1% sodium deoxycholate; and 0.1% SDS) containing 0.1% phenylmethane sulfonyl fluoride. Protein concentration of cell lysates was measured by BCA kit (Beyotime Institute of Biotechnology, Shanghai, China). Equal protein lysate samples (30 µg) were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membrane, which was blocked in 5% nonfat milk at room temperature for 2 h, followed by incubation with primary antibodies at 4°C overnight. The membrane was then incubated with secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG; catalog no. G-21234, dilution 1:5,000) or goat anti-mouse IgG (catalog no. G-21040, dilution 1:5,000) antibodies (Pierce; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. Blots were developed using an enhanced chemiluminescence kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The densitometric analysis of bands was performed using FluorChem SA (6.0.0) (Informer Technologies, Inc, Chicago, IL, USA).
5
Western Blot Analysis of Apoptosis Markers
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Western blot was executed as previously described [16 (link)]. Shortly, RIPA Lysis Buffer was adopted to extract total proteins. Later on, protein samples were loaded onto 10% SDS-PAGE gel and then transferred to a polyvinylidene fluoride (PVDF) membrane. Subsequently, the membrane was incubated with primary antibodies against Bax (ab243140, 1:500), Bcl-2 (ab32124, 1:1000), IGF1R (ab131476, 1:1000) and β-actin (ab6276, 1:3000) were procured from Abcam (Cambridge, MA, USA). In addition, secondary antibody (ab6702, 1:3000) was used for western blot analysis. Finally, enhanced chemiluminescence kit (Solarbio) was utilized to visualize the protein bands.
6
Western Blot Analysis of ADAR1, BDNF Proteins
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The protein samples of SH-SY5Y cells were extracted with protein extraction kit (Keygen Biotech, China). BCA protein detection kit (Keygen Biotech, China) was used to detect the protein concentration. The protein of each sample was 30 μg. After protein denaturation, the sample was added into 10% SDS polyacrylamide gels. The protein was transferred to polyvinyl difluoride membranes and blocked with 5% bovine serum albumin for 1 h (Beyotime, China). The membranes with the target protein were incubated with ADAR1 polyclonal antibody (1:1000, Proteintech, USA), BDNF polyclonal antibody (1:1,000, Bioss, China) and GAPDH monoclonal antibody (1:5000, Immunoway, USA) at 4 °C overnight. After that, the samples were washed with Tris-buffered saline containing Tween-20 (TBST), goat anti rabbit or goat anti mouse IgG (1:10000, Bioss, China) labeled with horseradish peroxidase was incubated with the membranes for 1 h at room temperature. Then the samples were washed with TBST, the target protein was detected by enhanced chemiluminescence Kit (Solar bio, China), and the optical density of each band was analyzed by image analysis system (Bio-Rad, USA), which was normalized to GAPDH protein level for data analysis.
7
Western Blot Protein Quantification
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Detailed operational steps for this procedure are outlined in our previous research.21 (link) The protein bands were visualized using an enhanced chemiluminescence kit (Solarbio, Beijing, China). Band intensities were quantitatively analyzed using the ImageJ software, and protein expression was normalized to β-actin signals.
8
Extracellular Vesicle Protein Profiling
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The samples including sEVs, HUVECs, and HDFs were homogenized in lysis buffer containing proteinase and phosphate kinase inhibitors (Solarbio, China). Proteins (20 µg) were mixed with loading buffer, electrophoresed via SDS‒PAGE gels, and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking in 5% nonfat milk, membranes were co‐incubated with primary antibodies against tumor susceptibility gene 101 (TSG101), heat shock protein (HSP70), CD9, GAPDH, Calnexin, PTEN, phospho‐ protein kinase B (p‐AKT), AKT, hypoxia‐inducible factor – 1α (HIF‐1α), vascular endothelial growth factor (VEGF), p21, p16, p53, Type I collagen (Col I), Type III collagen (Col III), α‐Smooth Muscle Actin (α‐SMA), and β‐actin (1:1000, CST, USA) at 4 °C overnight. Afterward, membranes were washed with Tris‐Buffered SalineTween‐20 (TBST) and co‐incubated with horseradish peroxidase‐conjugated anti‐mouse or anti‐rabbit IgG (1:5000, ZSGB‐BIO, China) for 1 h (RT). Bands were visualized by an enhanced chemiluminescence kit (Solarbio, China). The gray value assay representing the protein expression level was quantified by Image J.
9
Western Blot Analysis of TRAF6, JNK, and ERK
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Cells in different groups were, respectively, lysed with RIPA lysis buffer (Beyotime, Shanghai, China), and the supernatant was subsequently collected followed by centrifugation to extract the total protein, with its concentration probed by a BCA protein assay kit (Solarbio, Beijing, China). Notably, equal amount (20 μg) of protein samples in each group were separated by SDS-PAGE, and transferred to polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA), which was blocked with 5% skimmed milk for 1 h at ambient temperature, and specifically incubated with primary antibody at 4°C overnight. TRAF6 antibody (ab33915, 1:1000) and GAPDH antibody (ab181602, 1:1000) were purchased from Abcam (Shanghai, China). phospho-JNK antibody (p-JNK; #4668, 1:1000), phospho-ERK antibody (p-ERK; #4370, 1:1000), JNK antibody (#9252, 1:1000) and ERK1/2 antibody (# 4695, 1:1000) were available from Cell Signaling Technology (Danvers, MA, USA). Afterward, the membranes were incubated with goat anti-rabbit IgG (ab205718, 1:3000, Abcam, Shanghai, China) for 1 h at 37°C. Ultimately, the protein bands were developed by enhanced chemiluminescence kit (Solarbio, Beijing, China).
10
Retinal Protein Isolation and Western Blot Analysis
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Total proteins were isolated from retinal tissues and cells with RIPA buffer containing PMSF protease inhibitor (Solarbio). The concentration of proteins was measured with a BCA assay kit
(Solarbio). Western blot protocol was performed according to standard procedures. The antibodies included SOX9 (1:1,000, ABclonal), TXNIP (1:1,000; ABclonal), NLRP3 (1:1,000; ABclonal),
cleaved caspase-1 (1:500; Affinity, Cincinnati, OH, USA), and rabbit IgG (1:3,000, Solarbio). Visualization of the bands was performed using an enhanced chemiluminescence kit (Solarbio).
(Solarbio). Western blot protocol was performed according to standard procedures. The antibodies included SOX9 (1:1,000, ABclonal), TXNIP (1:1,000; ABclonal), NLRP3 (1:1,000; ABclonal),
cleaved caspase-1 (1:500; Affinity, Cincinnati, OH, USA), and rabbit IgG (1:3,000, Solarbio). Visualization of the bands was performed using an enhanced chemiluminescence kit (Solarbio).
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