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Both treated and untreated cells were washed twice with ice‐cold PBS and extracted on ice with cell lysis buffer (KeyGen Biotech Co. Ltd., Nanjing, China). The concentration of the protein was quantified using the BCA Protein Assay Kit (KeyGen Biotech Co. Ltd., Nanjing, China). Thirty micrograms of total protein was electrophoresed through 10% and 12% SDS PAGE and was then transferred to membranes (Millipore, USA). The membranes were blocked in 5% fat‐free milk for two hours then incubated with primary antibodies at 4°C overnight. Secondary antibodies were labelled with Horseradish Peroxidase (Abbkine Scientific, China) and the signals were detected using the ECL Kit (Advansta, UK). Subsequently, the images were analysed using the ImageJ 6.0 software. A glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) antibody was used as the control for whole‐cell lysates.
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Protein lysates from cells receiving different treatments were prepared using RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (KeyGEN, Nanjing, China). Equal amounts of total protein (30 μg of protein per lane) were loaded onto gels for electrophoresis and then transferred onto PVDF membranes (Millipore, Billerica, USA), followed by incubation in blocking buffer (25 mM Tris, pH 7.4, 0.15 M NaCl, 0.1% Tween-20, 5% nonfat milk) for 1 h at RT. The membranes were incubated with primary antibodies against α-smooth muscle actin (α-SMA) (Proteintech, Chicago, USA), IL-25 (R&D, Minneapolis, USA), total ERK1/2, phospho-ERK1/2, total AKT, phospho-AKT, phospho-B-Raf (Cell Signaling Technology, Boston, USA) or GADPH (Proteintech, Chicago, USA) at 4 °C overnight. After washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at RT. Proteins were visualized with an ECL kit (Advansta, CA, USA). GADPH was used as a loading control. The intensity of Western blotting bands was analyzed with Quantity One v4.6.2 software.
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Total proteins were extracted from the cells using RIPA buffer with protease inhibitors (Beyotime, Shanghai, China) on ice, subjected to SDS-PAGE and electrophoretically transferred to PVDF membranes (Merck Millipore, MA, USA). Membranes were incubated in 5% non fat milk dissolved in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 2h at room temperature and then incubated with primary antibodies as follows: EPB41L3 (1:500, Abcam, Cambridge, MA, USA), GAPDH (1:2000, Proteintech, Wuhan, China), followed by incubation with appropriate correlated HRP-conjugated secondary antibody. Then the membranes were incubated with secondary antibodies (Proteintech, Wuhan, China) at room temperature for 2h. Immunoblots were visualized by enhanced chemiluminescence (ECL kit, Advansta, USA) and scanned using ECLTM chemiluminescence detection system (Pierce, USA).
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The treated cells were lysed in an appropriate volume of RIPA lysis buffer (Servicebio, China) containing 1% phenylmethanesulfonyl fluoride (Servicebio, China) and placed on ice for 30 min. Next, the mixture was centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was collected and protein concentration was determined by the bicinchoninic acid assay (Beyotime Biotechnology, Shanghai, China). Then, 5x loading buffer (Beyotime Biotechnology, Shanghai, China) was added to the supernatant and heated at 100°C for 15 min. The proteins were separated on sodium dodecyl sulfate-polyacrylamide gels (Beyotime Biotechnology, Shanghai, China) and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore). The membrane was blocked in 5% skimmed milk at room temperature for 2 h and then incubated with primary antibody (β-actin antibody was diluted 1:4000, other antibodies (MMP-9, N-CAD, Twist, Vimentin and ZEB2) were diluted 1:1000) overnight at 4°C, followed by incubation with appropriate secondary antibody for 2 h at room temperature. Protein expression was detected using the ECL kit (Advansta, San Jose, CA, USA). The primary antibodies (β-actin, MMP-9, N-CAD, Twist, Vimentin and ZEB2) and the secondary antibody were purchased from Servicebio.
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Matured COCs (30 per sample) were lysed using PRO-PREP protein lysis buffer (iNtRON, Daejeon, Korea), and the lysates were separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. The separated protein bands were transferred onto nitrocellulose membranes (Pall Life Sciences, Port Washington, NY, USA), and the membranes were incubated with anti-cleaved caspase-3 (1:3000; #9664; Cell Signaling, MA, USA), anti-PARP1/cleaved PARP1 (1:4000; Santa Cruz, CA, USA), anti-AIF (1:3000; Santa Cruz), anti-Cyto C (Cyto C; 1:3000; Abcam, Cambridge, MA, USA), and anti-β-actin (1:3000; Santa Cruz) antibodies. The membranes were then probed with a secondary horseradish peroxidase (HRP)-conjugated anti-mouse/rabbit IgG (Thermo, Rockford, IL, USA) or an anti-goat IgG (AbFrontier, Seoul, Korea) secondary antibody overnight. Blots were developed using an enhanced chemiluminescence (ECL) kit (Advansta, Menlo Park, CA, USA) according to the manufacturer’s instructions. The amount of protein was calculated depending on band densities using Fusion Solo software (Vilber Lourmat, Collégien, France), and the bands were scanned using ImageJ software (NIH).
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Total protein was extracted from cells. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime, Jiangsu, China) and then transferred onto PVDF membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed at room temperature for 2 h. Anti-CLDN8, anti-IL22, anti-p-ERK, anti-ERK, anti-cleaved caspase-3, anti-bax, anti-bcl-2, anti-XIAP, anti-VEGF, anti-MMP-2, anti-E-cadherin, anti-N-cadherin and anti-GAPDH (1:800, abcam) were added overnight at 4 °C. The membranes were subsequently incubated with goat anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (1:5000, abcam) at room temperature for 2 h. Finally, proteins were visualized using a WestrenBright ECL Kit (Advansta, USA).
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Total proteins were extracted from differently treated HCC cells with RIPA buffer (Beyotime, Shanghai, China) on ice. The proteins were isolated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, German). Next, the membranes were incubated with 5% non–fat milk in Tris‐buffered saline for 1.5 hours at room temperature and then incubated with primary antibodies as follows: CyclinD1 (diluted 1:500; Bioworld Technology, Nanjing, China), MMP2 (diluted 1:500; Bioworld Technology, Nanjing, China), and MMP9 (diluted1:400; Bioworld Technology, Nanjing, China). The secondary antibodies applied HRP‐conjugated goat anti‐rabbit IgG (diluted 1:2000; Bioworld Technology, Nanjing, China) for 2 hours at room temperature. GAPDH was used as a loading control. The blot was visualized by enhanced chemiluminescence (ECL kit; Advansta) and scanned with an ECLTM chemiluminescence detection system (Pierce).
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Lipopolysaccharide (LPS) from E. coli 055: B5, LC–MS and HPLC-grade solvents and fine chemicals were procured from Sigma-Aldrich, USA. SYBR green mix cDNA synthesis kits were purchased from Takara Bioscience, India. ELISA kits (IL-6, CCL2, INF-γ and IL-1β) were obtained from R&D Systems. Primary antibodies were purchased from Cell Signaling Technology, USA, and the corresponding catalogue numbers are mentioned in supplementary table S2. Bicinchoninic acid reagent (BCA kit) was bought from Thermo Scientific, USA. Polyvinylidene difluoride (PVDF) membrane (0.45 μm) was procured from Millipore, USA. Secondary antibodies were procured from Jackson Laboratory, USA, and an ECL kit was purchased from Advansta, Menlo Park, CA, USA. Deionized water (18 MΩ) was used for the preparation of all solutions. Leaves of G. Sylvestre were collected from Chittoor district, Andhra Pradesh, India. The plant specimen was authenticated by a botany professor from Osmania University, Hyderabad, India. LC–MS CHROMASOLVs-grade and HPLC-grade methanol (MeOH) and acetonitrile (ACN) were procured from Sigma-Aldrich (St Louis, USA). Analytical reagent (AR)-grade ammonium acetate, ammonium formate, formic acid, hydrochloric acid (HCl).
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Cells were lysed with RIPA buffer (CW biotech, Beijing, China) and equivalent amounts of protein were separated by 10% SDS-PAGE and transferred to PVDF membranes. After being blocked in 5% nonfat milk in TBST for 1 h at room temperature, the membranes were incubated with the primary antibodies at 4°C overnight. Subsequently, the immunoblots were then incubated with a secondary antibody at room temperature. The membranes were developed using an electrochemiluminescence (ECL) kit (Advansta, USA) according to the manufacturer's protocol.
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After treating with the tested drugs, liver samples were lysed with RIPA buffer (CW biotech, Beijing, China). Nuclear extracts were prepared with a Nuclear Extract Kit (Thermo, USA). Equivalent amounts of protein were separated by 10% SDS-PAGE and transferred to PVDF membranes. After being blocked in 5% nonfat milk in TBST for 1 h at room temperature, the membranes were incubated with the primary antibodies at 4°C overnight. Subsequently, the immunoblots were incubated with a secondary antibody at room temperature. The membranes were developed using an electrochemiluminescence (ECL) kit (Advansta, USA).
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