Total proteins were extracted from HPSMCs in each treatment groups using a Total Protein Extraction Kit (cat. no. KGP2100; Nanjing KeyGen Biotech Co., Ltd.). Protein concentration was quantified using a bicinchoninic acid protein assay kit (Nanjing KeyGen Biotech Co., Ltd.). SDS-PAGE and immunoblotting were performed according to the manufacturer's protocols (Bio-Rad Laboratories, Inc.). Briefly, polyvinylidene fluoride membranes (EMD Millipore) were blocked using 5% skim milk dissolved in TBS (pH 7.5)-0.1% Tween-20 (TBS-T) for 2 h at RT after protein transfer from 12% SDS-PAGE gels (50 mg/lane). Subsequently, the membranes were incubated overnight at 4˚C with either the rabbit polyclonal anti-PDE5A3 antibody (cat. no. ab64179; 1:1,000; Abcam) or rabbit polyclonal anti-cGMP antibody (cat. no. ab12416; 1:2,000; Abcam). The membranes were then washed with TBS-T and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (1:5,000; cat. no. KGAA35; Nanjing KeyGen Biotech Co., Ltd.) at RT for 2 h. Enhanced Chemiluminescence Detection Kit (Bio-Rad Laboratories, Inc.) and the ImageJ software (version 1.5i; National Institutes of Health) were used to detect and semi-quantitatively analyze the immunoreactive proteins. This experiment was performed three times.
Enhanced chemiluminescence detection kit
Manufactured by Bio-Rad
Sourced in United States
The Enhanced chemiluminescence detection kit is a laboratory equipment designed for the detection and analysis of proteins in Western blotting applications. The kit provides a sensitive and reliable method for visualizing target proteins labeled with specific antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Sc 2039, by Santa Cruz Biotechnology (4 mentions)
Image station 2000r, by Kodak (4 mentions)
Goat anti mouse igg, by Santa Cruz Biotechnology (3 mentions)
μ drop reader, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Ripa buffer, by Cell Signaling Technology (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (3 mentions)
Mab8929, by R&D Systems (3 mentions)
Mab3925, by R&D Systems (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Image pro plus computer assisted image analysis system, by Media Cybernetics (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti tnf α, by Santa Cruz Biotechnology (2 mentions)
Af3169, by R&D Systems (2 mentions)
Sc 390991, by Santa Cruz Biotechnology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Cox 2, by Cell Signaling Technology (2 mentions)
51 protocols using enhanced chemiluminescence detection kit
1
Protein Extraction and Immunoblotting in HPSMCs
2
Quantifying SMN Isoforms in Duodenum
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Duodenum samples were homogenized using a Precellys Homogenizer (Berlin Technologies) in RLT lysis buffer using RNeasy Mini Kit (Qiagen, Chatsworth, CA). Total RNA extraction and reverse-transcription were performed as described previously [10 (link),31 (link)]. The ratio of full-length SMN2 to Δ7 SMN2 transcripts was quantified by quantitative real-time RT-PCR. The expression of human SMN protein was measured by western blotting as previously described [31 (link)]. β-tubulin was used as loading control in all samples. Blots were developed with an enhanced chemiluminescence detection kit (Bio-Rad, California, USA). Semi-quantification of band intensity was analyzed by imageJ software.
3
Nuclear Protein Extraction and Immunoblotting
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The nuclear protein extraction and western blot analysis were performed following the methods described by Almeer et al. [21 (link)]. The utilized antibodies included mouse anti-Nrf2 (sc-28379, 1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse antihistone H1 (sc-393358, 1 : 1500; Santa Cruz Biotechnology), and goat antimouse IgG (sc-2039, 1 : 5,000; Santa Cruz Biotechnology). Immunoblot analyses of antimouse IgG horseradish peroxidase-conjugated antibodies were performed using an enhanced chemiluminescence detection kit (Bio-Rad, USA). Data were acquired in arbitrary densitometric units using the ImageJ software.
4
Western Blot Analysis of Liver Proteins
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Western blot analysis was done as mentioned method (Kim et al., 2021b (link); Ko et al., 2020b (link)). The liver tissues were homogenized on chilled radioimmunoprecipitation assay buffer (Cell Signaling Technology, Danvers, MS, USA) with 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich Co.), and then centrifuged at 14,000 rpm for 30 min at 4°C. Protein contents were measured using a μ-drop reader (Thermo Fisher Scientific), and 30-μg protein was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to a nitrocellulose membrane. The primary and secondary antibodies used are as shown in Table 1 . After the reaction, enhanced chemiluminescence detection kit (Bio-Rad, Hercules, CA, USA) was used for blot membrane. Image-Pro plus computer-assisted image analysis system (Media Cybernetics Inc.) was used for detected bands. The control group was set to 1.00.
5
Western Blot Analysis of Glut1 Expression
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The treated macrophage cells were washed with ice-cold PBS and then lysed in RIPA cell lysis buffer, and 1% Halt protease inhibitor cocktail (Thermo Fisher). The resulting cell lysates were centrifuged for 20 min. at 20,000 g at 4°C, and the protein concentrations in the supernatants were determined using a Pierce BCA protein assay kit (Thermo Fisher) [24 (link)]. 20 μg proteins were loaded onto 10% SDS-PAGE gels, followed by electro-transfer onto nitrocellulose-membrane (Bio-Rad). The membranes were blocked in 1 × TBST (0.1% Tween-20, 20 mM Tris–Cl (pH 8.0), and 150 mM NaCl) containing 5% nonfat dry milk powder and then incubated with the primary antibodies against Glut1 (1:1000 dilution, 66290, Proteintech), GAPDH (1:1000 dilution, 60004, Proteintech), overnight at 4°C. Membranes were washed 3 times (1xTBST), and incubated with horseradish peroxidase-conjugated secondary antibodies (1:10000, SA00001, Proteintech) for 1 h at room temperature and then washed 3 times (1 × TBST). Lastly, immunoreactive proteins were detected using enhanced chemiluminescence detection kit (Bio-Rad). The band density was quantified by Image J 1.8.0172 software (National Institutes of Health) and the representative data were experiment normalized to nontreated control.
6
Quantifying STAT3 Phosphorylation in Cancer Cells
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Cancer cells were lysed with 1% SDS containing protease and phosphatase inhibitors (Sigma-Aldrich), and protein was quantified with the BCA Protein Assay Kit (Pierce). Proteins (10–20 µg) were resolved on 7.5% SDS-PAGE gels, transferred to polyvinylidene fluoride membranes (Millipore), blocked with 5% milk at room temperature for 2 h, stained with anti-human and murine pSTAT3 (Y705; Cell Signaling) or anti-human and murine tubulin (Santa Cruz Biotechnology) antibodies, and quantified using the enhanced chemiluminescence detection kit (Bio-Rad) and a LI-COR imager.
7
Evaluating Intracellular Signaling Pathways in UVB-Irradiated Cells
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To determine the effects of WESBD and EESBD on intracellular signaling pathways upregulated by UVB irradiation, the effects of the treatments on AKT and MAPK phosphorylation of the NIH-3T3 cells were evaluated by Western blotting. After cell lysis in lysis buffer [29 (link)] for 1 h. The cell lysates were centrifuged (13,000× g, 4 °C, 15 min). The protein concentration in the separated supernatant was measured using the Bradford assay [30 (link)]. For estimating the expression of each molecule, 20 µg of cell lysates were electrophoresed in sodium dodecyl sulfate polyacrylamide gel and then transferred to nitrocellulose membranes. After incubation with blocking buffer (5% w/v of nonfat milk in tris-buffered saline with Tween 20 (TBS-T) for 1 h), the membrane was reacted with a primary antibody (1:5000) for 16 h at 4 °C. After washing with TBS-T buffer three times, the membrane was reacted with a secondary antibody (1:3000) and conjugated with horseradish peroxidase (Santa Cruz, Dallas, TX, USA) at room temperature for 2 h. Each protein band was visualized using an enhanced chemiluminescence detection kit (Bio-Rad, Hercules, CA, USA).
8
Hippocampal Protein Extraction and Analysis
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Protein lysates were extracted according to a previously described method (Hwang et al. 2018 ; Kim et al. 2018 (link)). Dissected hippocampal tissues were homogenized in 400 μl per 1 g concentration of RIPA buffer (Cell Signaling Technology, Beverly, MA, USA) with 1 mM PMSF (Sigma-Aldrich Inc.) on ice. The homogenized sample was incubated on ice for 20 min. Incubated sample was centrifuged at 14,000 g for 10 min at 4°C, and supernatants were collected. Protein content was measured using a micro-drop plate reader (Thermo Fisher Scientific). NF-кB and IкBα in the hippocampus were detected using nuclear/cytosol fractionation kit (BioVision Inc, Milpitas, CA, USA) according to the manufacturer’s instructions. The following primary antibodies (1:1000 dilution) were selected to react overnight at 4°C: mouse anti-β-actin, anti-TNF-α, anti-interleukin-6 (IL-6), anti-proBDNF, anti-BDNF, anti-Iba-1, rabbit anti-TrkB, anti-NF-κB, and anti-IκBα (Santa Cruz Biotechnology). Subsequently, membranes were incubated for 1 h with attempt secondary antibodies (1:2000; Vector Laboratories). Blot membrane was detected using the HRP-conjugated IgG (Vector Laboratories) and the enhanced chemiluminescence detection kit (Bio-Rad, Hercules, CA, USA). Detected bands were quantified by Image-Pro® plus image analysis system (ver. 6.0, Media Cybernetics Inc., Silver Spring, MD, USA).
9
Western Blot Analysis of Liver Proteins
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Pieces from the liver were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing proteinase and phosphatase inhibitors. The protein concentration in the homogenates was determined using Bradford reagent [39 (link)] and 40 µg proteins were subjected to 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The separated proteins were transferred to nitrocellulose membranes which were blocked using 5% skimmed milk in tris buffered saline/tween 20 (TBST). After blocking, the membranes were probed with antibodies against pAkt Ser473, Akt, pGSK-3β Ser9, GSK-3β, and β-actin (Novus Biologicals, Centennial, CO, USA) overnight at 4 °C. The blots were washed three times with TBST and incubated with the secondary antibodies for 1 h at room temperature. The membranes were washed three times with TBST and developed using enhanced chemiluminescence detection kit (BIO-RAD, Hercules, CA, USA). The developed blots were scanned, and the band intensity was quantified using ImageJ (version 1.32j, NIH, USA).
10
Protein Expression Analysis Protocols
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Cell lysates were prepared with radioimmune precipitation buffer (1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, 150 mm NaCl, and 50 mm Tris, pH 7.5) containing protease and phosphatase inhibitor mixture solutions (GenDEPOT, Barker, NY). Thirty micrograms of total protein from each lysate was resolved on 5–12% SDS-polyacrylamide gels and electrotransferred to polyvinylidene fluoride (PVDF) membranes. After blocking in phosphate-buffered saline and 0.05% Tween 20 (PBST) containing 5% skim milk for 2 h, the membranes were incubated with the indicated primary antibodies overnight at 4 °C. Visualization of the immunoblots was performed using an enhanced chemiluminescence detection kit from Bio-Rad.
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