Total proteins were isolated from cells using RIPA buffer (Invitrogen; Thermo Fisher Scientific, Inc.), followed by BCA assay (Invitrogen; Thermo Fisher Scientific, Inc.) to measure protein concentrations. Protein samples were first denatured at 95°C for 10 min. Proteins (20 µg) were then separated by 8–15% SDS-PAGE. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes, followed by blocking in phosphate-buffered saline (PBS) containing 5% non-fat milk for 2 h. Membranes were first incubated with rabbit primary antibodies of JAK2 (product code ab39636; Abcam) and GAPDH (product code ab9485; Abcam) at 4°C for 15 h, followed by incubation at room temperature for 2 h with HRP IgG goat anti-rabbit (product code ab97051; Abcam). ECL solution (Sigma-Aldrich; Merck KGaA) was dropped onto membranes to develop signals. Enhanced chemiluminescence reagent (Amersham; Cytiva) was used to detect the signal on the membrane. The data were analyzed via densitometry using Image-Pro Plus software (64-bit Java 1.8.0_112; Media Cybernetics, Inc.) and normalized to expression of the internal control GAPDH.
Ecl solution
Manufactured by Merck Group
Sourced in United States, Germany, Switzerland
The ECL solution is a chemical reagent used in Western blot analysis to detect and quantify proteins. It produces a luminescent signal that can be captured and measured by specialized imaging equipment. The solution contains the necessary components to enable the chemiluminescent reaction, allowing for the visualization and analysis of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (31 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (14 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
β actin, by Merck Group (8 mentions)
Ab6721, by Abcam (6 mentions)
Ripa buffer, by Merck Group (6 mentions)
Trans blot turbo transfer system, by Bio-Rad (4 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Gapdh, by Merck Group (3 mentions)
Piercetm bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
β actin, by Abcam (3 mentions)
Nfatc1, by Santa Cruz Biotechnology (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
123 protocols using ecl solution
1
Western Blot Analysis of JAK2 and GAPDH
2
Comprehensive Protein Expression Analysis
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H9C2 cells or myocardial tissue were treated with RIPA lysis buffer containing protease and phosphatase inhibitors (Sangon Biotech, Shanghai, China), and total protein concentrations were measured using a BCA protein assay kit (Sangon Biotech, Shanghai, China). After denaturation at 95°C for 5 min in SDS sample buffer, proteins were separated on SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% (w/v) BSA for 1 h, followed by incubation overnight at 4°C with specific antibodies (all from Abcam, Cambridge, UK) against the following proteins: Bax (ab32503), BCL-2 (ab196495), pro-caspase 3(ab184787), cleaved-caspase 3 (ab214430), DRP-1 (ab184247), OPA-1 (ab42364), PGC-1 α (ab106814), SUCLA (ab202582), ACADL (ab129711), CD36 (ab133625), CPT-1 (ab234111), P-ACC (ab68191), ACC (ab45174), p-AMPK (ab133448), AMPK (ab207442), and GAPDH (ab181602). After washing, the membrane was incubated with an HRP-coupled secondary antibody for 60 min, washed again, and finally incubated with enhanced chemiluminescence (ECL) solution (Sigma, USA).
3
Immunoblotting Protocol for Phosphorylation Analysis
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For immunoblotting, proteins were separated by SDS–PAGE and then transferred to a nitrocellulose membrane (Optitran BA-S 85, pore size 0.45 µm, GE Healthcare) using a semi-dry blotting chamber at a constant current of 140 mA/gel for 60 min. After transfer, the membrane was blocked in 5% milk-powder in TBS-T for 1 h at room temperature or at 4°C overnight. Then, the membrane was incubated with primary antibody (pSer2 CTD 1 : 100, pSer5 CTD 1 : 500, pSer7 CTD 1 : 100, pThr4 CTD 1 : 100, thiophospho 1 : 5000). Afterwards, the blot was washed 3 × 5 min in TBS-T and was then incubated with an appropriate secondary antibody (1 : 5000) for 1 h. Membrane was again washed 3 × 5 min in TBS-T. For detection, membranes were immersed with ECL-solution (Sigma Aldrich) for 1 min and then analysed with a CCD camera in a XRSChemDoc system (BioRad).
4
Survivin expression analysis by Western blot
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The effects of HCP5 and miR-320 overexpression on the expression of survivin were analyzed by Western blot. H23 cells were harvested at 48h post-transfection and cells were counted. Total proteins were extracted using RIPA solution (Sangon). To denature proteins, all protein samples were incubated in boiling water for 10 min. Twelve percent SDS-PAGE gel was used to perform electrophoresis, followed by gel transfer to PVDF membranes. Membranes were first incubated with primary antibodies of rabbit anti-survivin (1:2000, ab469, Abcam) and anti-GAPDH (1:1500, ab37168, Abcam), and the incubation was performed at 4°C for 18h. The secondary antibody was goat HRP (IgG) (1:1800; ab6721; Abcam) and the incubation was performed at 24°C for 2h. ECL solution (Sigma-Aldrich) was used to develop signals. Image J v1.46 software was used to normalize signals.
5
Western Blot Analysis of p53 and GAPDH
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HS 722.T and RKO cell pellets were resuspended in RIPA solution (Sangon, Shanghai, China) at a concentration of 1 ml RIPA solution per 105 cells. Protein samples were denatured and subjected to 12% SDS-PAGE gel electrophoresis. Proteins were transferred to PVDF membranes. After blocking (5% non-fat milk for 2 h at 25ºC), rabbit polyclonal primary antibodies of GAPDH (1: 1300, ab181602, Abcam) and p53 (1: 1300, ab131442, Abcam) were used to incubate membranes at 4°C for at least 12 h. After that, goat anti-rabbit lgG-HRP secondary antibody (1: 1000, MBS435036, MyBioSource) was used to further incubate with membranes for 2 h at 25°C. Signals were developed by incubating with ECL solution (Sigma-Aldrich, USA) for 15 min at room temperature, and data were processed using Image J 1.48 software.
6
Western Blot Analysis of Apoptosis Regulators
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The cells were washed and scraped with PBS, and incubated in an RIPA buffer containing a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). After protein preparation, Bradford assay was performed. The same amounts of total protein (20 μg) were resolved in 12% sodium dodecyl sulfate (SDS)-acrylamide gel and transferred to the PVDF membrane. The following primary antibodies were used: PUMA, JNK, p- JNK, Caspase-3, −9, Bax and Bcl-2 1:3000 in 2% BSA (Cell signaling, Danvers, MA). β-actin (Santa Cruz Biotechnology, Inc., Dallas, TX) was used as the internal control. Membrane was incubated with the secondary antibody (1:10,000 dilution); the blot was detected with an ECL solution (EMD Millipore Corporation, Billerica, MA) using a Davinch-Chemi™ chemiluminescence imaging system (Davinch-K, Seoul, South Korea).
7
Protein Expression Analysis in MC3T3-E1 Cells
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Total protein was extracted from MC3T3-E1 cells on the Ti discs using a NP-40 lysis buffer. After electrophoresis and transferring protein, the membrane was blotted with the primary antibodies for 16 h at 4°C, such as 1:2000 of anti-rabbit Runx-2 (SantaCruz Biotechnology Inc., Dallas, TX, USA), 1:2000 of anti-goat OPG (SantaCruz Biotechnology Inc.), 1:1000 of anti-mouse RANKL (Novus Biological Inc., Centennial, CO, USA) and 1:2,500 of anti-mouse β-actin (Santa Cruz Biotechnology Inc.). After washing, the membrane was blotted with 1:5000 of either horseradish peroxidase (HRP)-conjugated goat anti-rabbit or mouse-IgG (Enzo Life Sciences Inc., New York, NY, USA) and HRP- conjugated donkey anti-goat-IgG (SantaCruz Biotechnology Inc.). The developing was performed using X-ray film (Fuji Film Co.) after detection using an ECL solution (Merck Millipore, Burlington, MA, USA). The density of the expressed bands was measured using Science Lab Image Gauge (Fuji Film Co.).
8
Western Blot Analysis of Protein Expression
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Total protein content was extracted from the cells using a RIPA lysis buffer containing PMSF (Beyotime, Beijing, China), and the protein concentration was determined using bicinchoninic acid (BCA) protein quantification kits (Wuhan Boster Biological Technology, Ltd., Wuhan, China). Subsequently, the obtained proteins were separated with 10% SDS–PAGE, then electrotransferred to poly(vinylidene fluoride) (PVDF) membranes, and blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature to block the non-specific binding. Afterward, diluted primary antibodies rabbit anti-mouse RBP-J (ab180588, dilution ratio of 1:1000, Abcam), CYLD (ab137524, dilution ratio of 1:1000, Abcam), p-p65 (ab194726, dilution ratio of 1:500, Abcam), p-IκBα (ab133462, dilution ratio of 1:1000, Abcam), and β-actin (ab8227, dilution ratio of 1:2000, Abcam) were added, respectively, and incubated overnight at 4°C. The following day, the membrane was washed and incubated with the goat anti-rabbit secondary antibody IgG (ab205718, dilution ratio of 1:2000, Abcam) for 1 h at room temperature and then developed with the ECL solution (EMD Millipore, United States). The grayscale of bands in the Western blot images was quantified using the Image Pro Plus 6.0 software (Media Cybernetics, United States), with β-actin serving as an internal reference.
9
Western Blotting Protocol
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Western blotting was performed according to the methods described in our previous study [50 (link)]. In brief, the lysates were collected, boiled with 5X sample buffer, and separated by SDS-PAGE. Proteins on the membrane were probed with specific antibodies, and the antibodies were detected by enhanced chemiluminescence (ECL) solution (EMD Millipore, Darmstadt, Germany). Uncropped images for the blots are provided in Figure S6 .
10
In Vitro Cellular Assays for Drug Evaluation
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GJ powder provided by Hanpoong Pharmaceutical Co. (Jeonju, Korea) was dissolved in D.W. LY294002, SC79, 3-methyladenine (3-MA), 3-[4,5-dimetylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) and cisplatin were purchased from Sigma-Aldrich (St. Louis, MO, USA), and chloroquine (CQ) was from Invitrogen (San Diego, CA, USA). ECL solution were obtained from Merck Millipore (Middlesex, MA, USA) and Z-VAD-FMK was provided by R&D Systems, Inc. (Northeast, MN, USA), Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s modified Eagle medium (DMEM), Roswell Park Memorial Institute 1640 (RPMI1640), penicillin and streptomycin were obtained from WELGENE (Gyeongsan, Korea). Fetal bovine serum (FBS) was obtained from GR scientific (Bedford, UK).
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