The primary antibodies, including anti-caspase-3, anti-caspase-9, anti-Nrf2, anti-NQO1, anti-HO-1, anti-NF-κB p65, and anti-actin, were purchased from Abcam (Cambridge, UK). After 24 h of reperfusion, the rat brain samples were homogenized, washed with phosphate buffered saline (PBS), and then lysed with radio immunoprecipitation assay (RIPA) lysis buffer. The total protein content was determined using a BCA kit (Solarbio, Beijing, China). After centrifugation at 12,000 rpm for 10 min, the supernatants were harvested for the measurement of protein concentration. The protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Solarbio, Beijing, China) and then transferred to polyvinylidene difluoride (PVDF) membranes (Solarbio, Beijing, China). The membranes were blocked with 5% nonfat dry milk and incubated overnight at 4 °C with anti-caspase-3, anti-caspase-9, anti-Nrf2, anti-NQO1, anti-HO-1, anti-NF-κB p65, and anti-actin. Following that, the membranes were washed with tris-buffered saline and Tween 20 (TBST) buffer. The secondary antibody was added to the membranes. The blots were detected with an ECL detection kit (Solarbio, Beijing, China). Actin was served as a loading control.
Ecl detection kit
Manufactured by Solarbio
Sourced in China, United States
The ECL detection kit is a laboratory equipment used for enhanced chemiluminescent (ECL) detection. It provides a sensitive and reliable method for detecting and quantifying proteins in western blot analyses.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Polyvinylidene fluoride membrane, by Beyotime (3 mentions)
Nuclear and cytoplasmic isolation kit, by Keygen Biotech (2 mentions)
Ab22555, by Abcam (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Ab97080, by Abcam (2 mentions)
Bca kit, by Solarbio (1 mentions)
Pvdf membrane, by Solarbio (1 mentions)
Anti actin, by Abcam (1 mentions)
Sds page, by Solarbio (1 mentions)
Anti caspase 3, by Abcam (1 mentions)
Anti nrf2, by Abcam (1 mentions)
Anti nqo1, by Abcam (1 mentions)
Anti ho 1, by Abcam (1 mentions)
Anti caspase 9, by Abcam (1 mentions)
Anti nf κb p65, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Gapdh antibody, by Proteintech (1 mentions)
Notch1, by Santa Cruz Biotechnology (1 mentions)
Bca kit, by Tiangen Biotech (1 mentions)
12 protocols using ecl detection kit
1
Protein Expression Analysis in Rat Brain
2
Quantitative Protein Analysis via Western Blot
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The cells under the described treatments were lysed in RAPI (protein lysis buffer containing protease inhibitor and phosphatase inhibitor) for 30 min on ice. The obtained proteins in the supernatants were then quantified using a BCA kit (Tiangen, Beijing, China). Equal amounts of protein sample (50 µg) were separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis, thus electrically transferring the proteins onto polyvinylidene fluoride membranes (Millipore, Boston, MA). Then, the resultant membranes were blocked with 5% skim milk in TBST buffer for 1 h at room temperature prior to coincubation with the corresponding primary antibodies against TIMP1 and TIMP2 (Proteintech, Wuhan, China) or Notch-1 (Santa Cruz Biotechnology) overnight at 4 °C. Subsequently, the membranes were incubated with secondary antibody (Jackson, West Grove, USA) for 1 h at room temperature. The gray levels of proteins were visualized with horseradish peroxidase-conjugated secondary antibodies via exposure to a chemiluminescence (ECL) detection kit (Solarbio, Beijing, China). The images were obtained using Quality One Imaging software (Bio-Rad, USA). The monoclonal GAPDH antibody (Proteintech, Wuhan, China) served as an internal control.
3
Western Blot Analysis of Protein Expression
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Radioimmunoprecipitation assay buffer (Solarbio, Beijing, China) was employed for protein extraction from cells. Protein concentration was estimated using a bicinchoninic acid assay kit (Beyotime, Shanghai, China). Protein samples were resolved in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto polyvinylidene fluoride membranes (Beyotime), which were then blocked with 5% defatted milk, incubated with anti-GAPDH (ab9485, 1:2500, Abcam), anti-GBA2 (ab205064, 1:500), and anti-ST3GAL5 (ab155671, 1:1000) primary antibodies (Abcam, Shanghai, China) at 4°C overnight, and washed thrice with Tris-buffered saline in Tween before incubation for 2 hours with the secondary antibody (ab205718, 1:2000, Abcam). Lastly, blot signaling was detected with an ECL detection kit (Solarbio) and estimated by ImageJ software (NIH, Bethesda, Md, USA).
4
Subcellular Fractionation and Western Blot Analysis
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Total proteins were extracted with RIPA buffer (Thermo Scientific, USA) and 1 mM PMSF. Cytoplasmic and nuclear subcellular fractionation was performed according to the manufacturer’s instructions using the Nuclear and Cytoplasmic Isolation Kit (KeyGEN, China). All proteins were separated on 10% SDS-PAGE (Invitrogen, USA) gels, transferred onto PVDF membranes (Millipore, USA), and then blocked using 5% skim milk. The proteins were detected by incubating the following primary antibodies: anti-AR (AR441; Abcam, USA), anti-estrogen receptor (ER; D8H8; CST, USA), anti-progesterone receptor (PR; 6A1; CST, USA), anti-HSP27 (G3.1, Abcam, USA), anti-HSP27 (phospho S15) (EP2293Y, Abcam, USA), anti-HSP27 (phospho S78) (Y175, Abcam, USA), anti-HSP27 (phospho S82) (EPR7278, Abcam, USA), anti-β-actin (8H10D10, CST, USA), and anti-Histone H3 (D18C8, CST, USA) overnight; and incubated with horseradish peroxidase-labeled anti-rabbit or anti-mouse IgG, followed by detection using the ECL detection kit (Solarbio, China). Each sample was analyzed in triplicate.
5
Quantitative Western Blot Analysis
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Total protein was isolated from LO2 cells or liver tissues with RIPA lysis buffer. Protein concentration was quantitated with the BCA Protein Assay Kit (Beyotime, P0012, China). 30 μg protein was separated in 10% SDS-PAGE gel and transferred to the PVDF membrane. After blocking with 5% bovine serum albumin (BSA), the membrane was incubated with primary antibody at 4 °C overnight followed by washing with TBST. HRP-conjugated secondary antibody incubation was performed at room temperature (RT) for 1 h. The signals were developed with the ECL detection kit (Solarbio, cat# PE0010, China) and detected by the ChemiScope 600 Exp system (ClinX, China). Antibodies used in the western blot include rabbit anti-FATP2 (1:1000, Proteintech, cat# 14,048–1-AP, USA), rabbit anti-GAPDH (CST, cat# 1574, USA), and HRP-conjugated goat anti-rabbit IgG (ZSGB-Bio, cat# ZB2301, China). The gray value of the protein band was quantitated with ImageLab 6.0 software (Bio-Rad, USA).
6
Protein Extraction and Western Blot
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Cellular protein was extracted by RIPA buffer containing PMSF. The Nuclear and Cytoplasmic Isolation Kit (KeyGEN, China) was used for the cytoplasmic and nuclear protein extraction according to the manufacturer’s instructions. The proteins were separated on 10% SDS-PAGE gels, transferred onto PVDF membranes, blocked using 5% skim milk, and incubated with primary antibodies overnight at 4 °C. The protein bands were detected by the ECL detection Kit (Solarbio, China).
7
Western Blot Analysis of Cellular Proteins
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Total tissue lysates were separated on SDS-PAGE and electrotransferred onto polyvinylidene fluoride membranes (0.22 μm) by wet blotting (Bio-Rad). Primary antibodies detected the following proteins: β-actin (1:100,000, Cat No. AC026, Abclonal), α-tubulin (1:50,000, Cat No. 66031-1-Ig, Proteintech), CX3CL1 (1:1000, Cat No. A14198, Abclonal), GNB5 (1:1000, Cat No. A4447, Abclonal), AKT2 (1:5000, Cat No. ab131168, Abcam), p-AKT (1:1000, Cat No. YP0006, Immunoway), NF-κB (1:2000, Cat No. 66535-1-Ig, Proteintech), p-NF-κB (1:1000, Cat No. 93H1, Cell Signaling Technology), Bad (1:1000, Cat No. ab32445, Abcam), Bcl-2 (1:2000, Cat No. ab182858, Abcam), occludin (1:3500, Cat No. 13409-1-AP, Proteintech), claudin-1 (1:4500, Cat No. 13050-1-AP, Proteintech), which were then incubated with goat anti-rabbit IgG and goat anti-mouse IgG and HRP-conjugated(1:5000, Cat No. CW0103S, CW0102S, CWBIO). Then, stripping buffer (CW0056M, CWBIO) was used for the replacement of antibodies. The density of each protein was detected by an ECL detection kit (Solarbio).
8
Western Blot Analysis of Histone Modifications
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Protein samples from PBMCs were homogenized in lysis buffer (50 mM Tris HCl, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton X-100, 0.3% NP-40, 0.1 mM PMSF and 1 µg/mL pepstatin A). Equal amounts of proteins (20 μg) were separated on 10% SDS–PAGE (Invitrogen, USA) gels, transferred onto PVDF membranes (Millipore, USA), and then blocked with 5% skim milk at room temperature for 2 h. The proteins were incubated with the following antibodies for overnight, washed with PBS, then were incubated with horseradish peroxidase-labeled secondary anti-mouse or anti-rabbit antibodies (1:2000) (ZSGB-Bio, Beijing, China) for 1 h at room temperature. Target signals were detected using the ECL detection kit (Solarbio, China). The intensities of blots were quantified by ImageJ software and relative protein levels were normalized by H3. Commercial antibodies used were as follows: anti-PanKcr (1:1000, #501), H2BK11cr (1:2000, #508), H2BK12cr (1:2000, #509), H2BK20cr (1:2000, #512) and H2BK34cr (1:2000, #514) from PTMbio, China, and anti-H3 (1:2000) from CST, USA.
9
Western Blot Analysis of AJAP1 and Ezrin
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Total proteins were extracted by using RIPA with PMSF according to the manufacturer’s protocol. An equal amount (30 µg) of samples was separated on 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Burlington, MA, USA). Then, the membranes were blocked in 5% non-fat milk for 1 h. Eventually, these membranes were detected using the ECL detection Kit (Solarbio, Beijing, China) after incubating the primary antibodies including anti-AJAP1 (AJAP1; Abcam; ab205496, rabbit secondary antibody) and anti-Ezrin (Ezrin; Santa Cruz Biotechnology, sc-58758, mouse secondary antibody).
10
Western Blot Analysis of Signaling Proteins
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Proteins were extracted from cells using Radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Jiangsu, China) and quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The extracted proteins were then separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), which were blocked using 5% skim milk. The PVDF membranes were then incubated with primary antibodies recognizing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000, CST, Danvers, MA, USA), AKT (1:1000, CST), phosphorylated (p)-AKT (1:1000, CST), PI3K (1:1000, CST), and p-PI3K (1:1000, CST) antibodies at 4 °C overnight. Next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Beyotime, Jiangsu, China). The immunoreactive protein bands were visualized using a chemiluminescence (ECL) detection kit (Solarbio, Beijing, China) and imaged using the ChemiDoc™ XRS imaging system (Bio-Rad, Hercules, CA,USA).
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