After homogenization of the exosomes and hippocampus, precooled RIPA (Thermo, 89900, USA) lysate was added and lysed on ice for 30 min. The supernatant was collected, and the concentration was measured by a BCA protein quantification kit (Thermo, 23225, USA). Briefly, 40 μg of proteins in the supernatant was separated by SDS-PAGE and transferred onto a PVDF membrane. CD9 (Abcam, ab92726, UK), CD63 (Abcam, ab59479,), and Calnexin (Abcam, ab22595,) primary antibodies were added to the exosomal tissue, and TLR2, TLR4, MyD88, and NF-κB were added to the hippocampus. Bcl 2 (Abcam, ab59348), Bax (Abcam, ab32503), pro-caspase-3 (Abcam, ab32150), and cleaved caspase-3 (Abcam, ab49822) primary antibodies were added onto the PVDF membrane and incubated overnight at 4°C. After washing with PBS, secondary antibodies were added and incubated at room temperature for 2 h. The ECL luminescence kit (Thermo Scientific, USA) was used to develop the color. Images were obtained by the Azure c600 Western Blot Imaging System (Azure, USA), and the gray values were read by Quantity One software.
Ecl luminescence kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ECL luminescence kit is a laboratory product designed to detect and measure the presence of specific proteins or molecules in a sample. It utilizes a chemical reaction that produces luminescence, which can be quantified and used for analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ipvh00010, by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Bca protein quantification kit, by Thermo Fisher Scientific (1 mentions)
Azure c600 western blot imaging system, by Azure Biosystems (1 mentions)
Goat anti rabbit igg h l hrp, by Beyotime (1 mentions)
Goat anti mouse igg h l hrp, by Beyotime (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ab238135, by Abcam (1 mentions)
Ab187041, by Abcam (1 mentions)
Ripa cell lysate, by Beyotime (1 mentions)
Bs 8845r, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg secondary antibody, by ZSGB-BIO (1 mentions)
Ab108319, by Abcam (1 mentions)
Imagej software, by Media Cybernetics (1 mentions)
Ab207802, by Abcam (1 mentions)
Ab219800, by Abcam (1 mentions)
Ab17942, by Abcam (1 mentions)
Ab223500, by Abcam (1 mentions)
10 protocols using ecl luminescence kit
1
Exosomal and Hippocampal Protein Analysis
2
Protein Extraction and Western Blot Analysis
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RIPA lysis buffer (Thermo Fisher) was applied to extract proteins from cells. Western blot assay was performed with reference to previous report (He et al., 2021 (link)). The antibodies used were: PDK4 (1:7000; Proteintech, 12949-1-AP), GAPDH (1:15000; Proteintech, 60004-1-Ig), Goat Anti-Mouse IgG H&L(HRP) (1:1000; Beyotime, A0216), Goat Anti-Rabbit IgG H&L(HRP) (1:1000; Beyotime, A208). Western blots were detected by ECL luminescence kit (Thermo Fisher) and visualized by chemiluminescence apparatus. ImageJ software was used for quantification.
3
Hippocampal Protein Analysis by Western Blot
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Hippocampal tissues were homogenized in RIPA cell lysate (Beyotime, P0013B), centrifuged at 12,000 × g for 15 min, and the supernatant was collected. Each protein sample was loaded into the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel spiked wells. Electrophoresis was performed at constant pressure of 80 V for approximately 1 h. The proteins were transferred to a polyvinylidene fluoride membrane (Millipore, IPVH00010), fixed with western closure solution (5% skim milk powder), slowly shaken on a shaker for 2 h at room temperature, and then incubated with the following primary antibodies: rabbit anti-postsynaptic density-95 (PSD-95) antibody (1:2000, abcam, ab238135), rabbit anti-synaptophysin (SYN) antibody (1:1000, Bioss, bs-8845R), rabbit anti-BDNF antibody (1:1000, abcam, ab108319), and rabbit tyrosine kinase receptor B (TrkB) antibody (1:5000, abcam, ab187041). The samples were further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:20000, Zsbio, ZB-2301) for 1.2 h. The membranes were washed with phosphate-buffered saline with Tween and the ECL luminescence kit (Thermo, 340,958) was used to detect the proteins. Finally, the intensity of the bands was analyzed by Image J software (Media Cybernetics, United States).
4
Western Blot Analysis of Apoptosis Markers
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RIPA (GBCBIO, G3424) was used to lyse cells and extract total protein to prepare protein samples. The protein lysates were separated by conventional electrophoresis, transferred onto the membrane, and washed with PBS, followed by blocking with 1% BSA for 2 h. Then, primary antibodies Box (ab32503, Abcam, Cambridge, UK), Bcl-2 (ab32124, Abcam, Cambridge, UK), Caspase-1 (ab32503, Abcam, Cambridge, UK), Cleaved Caspase-3 (ab32503, Abcam, Cambridge, UK), Caspase-8 (ab207802, Abcam, Cambridge, UK), Caspase-9 (ab2302, Abcam, Cambridge, UK), Caspase-11 (ab25901, Abcam, Cambridge, UK), Cyclin D1 (ab202068, Abcam, Cambridge, UK), ERK 1/2 (ab180673, Abcam, Cambridge, UK), p-ERK 1/2 (ab16663, Abcam, Cambridge, UK), WNT (ab17942, Abcam, Cambridge, UK), β-catenin (ab223500, Abcam, Cambridge, UK), GSDMD (ab15251, Abcam, Cambridge, UK) and β-actin (ab32572, Abcam, Cambridge, UK) (abcam, ab32503, ab32124, ab207802, ab2302, ab25901, ab202068, ab180673, ab16663, ab17942, ab223500, ab15251, ab32572, ab219800, ab8227) each at 1:1000 dilution were incubated with the membrane overnight at 4°C. The next day, HRP (ab32572, Abcam, Cambridge, UK) and labeled IgG secondary antibody (ab150077, Abcam, Cambridge, UK) each at 1:5,000 were further incubated with the membrane at room temperature for 1 h. ECL Luminescence Kit (Thermo, 32209) was used to develop the signal in a dark room.
5
Comprehensive Cell Analysis Toolkit
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TRIzol Extraction kit, RIPA, BCA Protein kit, Lipofectamine™ 2000, ECL Luminescence kit, Transwell kit (nos. 15596018, 89900, 23225, 11668030, 35055, A1142801; Thermo Fisher Scientific, Inc., Shanghai, China); H2O2, FBS, DMEM, Penicillin-streptomycin double-antibody (216763, F2442, D5796, V900929; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany); RT-qPCR kit, TransScript Green miRNA Two-Step RT-qPCR SuperMix (AQ202-01; Beijing TransGen Biotech Co., Ltd., Beijing, China), FN-1 monoclonal antibody, β-actin monoclonal antibody, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody (nos. MAB19182, MAB8929, HAF007; R&D Systems, Inc., Minneapolis, MN, USA), CCK-8 kit (C0037; Beyotime Institute of Biotechnology, Shanghai, China), miR-144-3p primer sequence, inhibition and over-expression sequences, and independence sequence were designed and produced by Sangon Biotech Co., Ltd. (Shanghai, China).
6
Protein Expression Analysis by Western Blot
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Protein was isolated from tissue using RIPA buffer. A NanoDrop 2000 (Thermo, USA) was used to measure the protein concentration. The protein samples were separated using SDS‒PAGE. After the protein was transferred to a PVDF membrane, a 3-h incubation in 5% skim milk at room temperature was performed to block nonspecific protein sites. Then, primary antibody was incubated with the membrane overnight. The PVDF membrane was then treated with the secondary antibody. Finally, an ECL luminescence kit (Thermo Scientific, USA) was used to visualize the protein on the PVDF membrane and capture images. Three separate experiments were performed, and the protein expression was analyzed using ImageJ each time. SERPIAN5, OLR1, PDGFA, S100A4 and APOH primary rabbit antibody were purchased from Abcam (Cambridge, USA), MSX1 primary rabbit antibody were purchased from Bioss (Beijing, China), secondary antibody (goat anti-rabbit immunoglobulin) were purchased from Abcam (Cambridge, USA).
7
Immunoblotting Analysis of Cartilage Tissue
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The cartilage tissue samples were collected and lysed by adding RIPA cell lysis solution (Beyotime, Shanghai, China). The supernatant was collected by centrifugation. The gels were then prepared, electrophoresed, and transferred to PVDF membranes (Millipore, MA, USA) before being poured into a closure solution and left for 2 h. Primary antibodies were diluted with closure solution as follows: TLR4 rabbit antibody (Bioss, Beijing, China), 1:1500; MyD88 mouse antibody (Santa Cruz, CA, USA), 1:1000; NF-κB p65 mouse antibody (Proteintech, IL, USA), 1:2000; p-NF-κB p65 rabbit antibody (CST, MA, USA); 1:1000. The PVDF membranes were incubated at 4 °C for 12 h. After completion of this step, a washing solution PBST (Zs-BIO, Beijing, China) was added. The horseradish peroxidase–labeled secondary antibody (Zs-BIO) was then diluted with secondary antibody dilution at 1:20,000 and incubated at room temperature for 2 h. The protein was detected using an ECL luminescence kit (Thermo Fisher Scientific, MA, USA), and the protein expression was determined by film strip analysis using ImageJ software.
8
Western Blotting for Hippocampal Protein Analysis
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The protocol of western blotting (WB) was performed as previously described (Wei et al., 2022 (link)). In brief, the hippocampal tissue was lysed in RIPA lysis buffer (Beyotime, P0013B) and centrifuged at 12,000 × g for 15 min to collect the supernatant. The protein samples were electrophoretically separated and then blotted onto polyvinylidene fluoride membranes (Millipore, IPVH00010). The protein levels were determined via incubation with antibodies against PGC‐1α (1:1000; abcam), SIRT1 (1:500; Santa Cruz), NF‐κB (1:5000; abcam), brain‐derived neurotrophic factor (BDNF; 1:1000; abcam), PSD‐95 (1:2000; abcam), SYN (1:1000; bioss), IL‐1β (1:500; bioss), IL‐6 (1:1000; Wanleibio), and TNF‐α (1:1000; bioss). The membranes were then incubated with horseradish peroxidase‐labeled secondary antibodies (1:20,000; Zsbio) according to the properties of the primary antibody. Protein detection was performed using an ECL luminescence kit (Thermo, 340958) and quantified with ImageJ software.
9
Quantifying RhoA and ROCK1 Levels
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RhoA and ROCK1 levels were examined by western blot.Tumor tissues or Huh-7 cells were used for western blot. Whole proteins were purified using a protein extraction kit (Promega, Beijing, China). After quantified, 30 µg whole protein was loaded on sodium dodecyl by BCA method sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred onto a polyvinylidene difluoride (PVDF) membrane, and then blocked at skim milk solution. Anti-RhoA (ab187027, Abcam) and anti-ROCK1 (ab134181, Abcam) antibodies were added and incubated overnight at 4 ℃. Diluted secondary antibodies (1:1,000, Abcam) were aliquoted and incubated for 2 hours. After detection by enhanced chemiluminescence (ECL) luminescence kit (Thermo Fisher Scientific, Madison, WI, USA), the protein blots were imaged in a gel imager and photographed. The grey scale values were calculated by Image J software [National Institutes of Health (NIH) image software].
10
Quantification of Neuronal Proteins
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The total proteins of cultured neurons were obtained using RIPA (Thermo, USA) to lysate and measured using the BSA kit (TaKaRa, Dalian). Next, aliquots containing 20 μg of protein were electrophoresed on 10% SDS-PAGE and transferred onto PVDF membranes (ThermoScientific, Madison, WI, USA). The PVDF membranes were blocked with TBST solution containing 5% skim milk at room temperature for 3 h and incubated with primary antibodies (including anti-KLK6, anti-PSD95, and anti-YN1-specific antibodies; the dilution ratio was 1:1000) overnight at 4 °C and with HRP-conjugated secondary antibody (1:10,000) at room temperature for 1 h. The protein bands were detected using a high-sensitivity ECL luminescence kit (ThermoScientific, Madison, WI, USA) and imaged using a chemiluminescence imaging analysis system.
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