Cells were washed with ice-cold phosphate-buffered saline (PBS, pH 7.4) and lysed in ice-cold HNTG buffer (50 mmol/L HEPES (pH = 7), 50 mmol/L NaF, 1 mmol/L EGTA, 150 mmol/L NaCl, 1% Triton X-100, 10% glycerol, 1.5 mmol/L MgCl2) containing a mixture of protease inhibitors (Roche Applied Science, Indianapolis, IN, USA). Proteins were separated by SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane. The membrane was blocked in TBST (20 mM Tris/HCl, pH 7.5, 500 mM NaCl and 0.1% Tween 20) containing 5% (w/v) non-fat dried milk for 1 h at room temperature. Proteins of interest were probed with corresponding primary antibodies followed by HRP (horseradish peroxidase)-conjugated anti-rabbit or anti-mouse secondary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection reagents (Applygen Technologies, Beijing, China) and Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA), respectively. For cell signaling study, cells were transfected with indicated plasmids and cultured for 36 h. Then the cells were starved in serum-free medium for 16 h before stimulation for 15 min with 10% FBS.
Ecl detection reagent
Manufactured by Applygen
Sourced in China
ECL detection reagents are a type of laboratory equipment used to detect and quantify the presence of specific proteins or molecules in a sample. They utilize the principles of enhanced chemiluminescence (ECL) to generate a luminescent signal, which can then be measured and analyzed. The core function of ECL detection reagents is to provide a sensitive and reliable method for protein detection and quantification in various applications, such as Western blotting, ELISA, and protein array analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (1 mentions)
A8717, by Merck Group (1 mentions)
Pvdf membrane, by Boster Bio (1 mentions)
Rabbit anti β actin, by Bioworld Technology (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Boster Bio (1 mentions)
Rnatrip reagent, by Applygen (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor, by Selleck Chemicals (1 mentions)
Anti lc3b, by Merck Group (1 mentions)
Anti pink1, by Abcam (1 mentions)
Anti tom20, by Abcam (1 mentions)
Anti α actin, by Abcam (1 mentions)
Bca reagent kit, by Applygen (1 mentions)
Anti p62 sqstm1, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Pierce bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Tris glycine precast gel, by Bio-Rad (1 mentions)
8 protocols using ecl detection reagent
1
Western Blot Analysis of Signaling Pathways
2
Detergent-soluble Aβ ELISA and Western Blot
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The extracts for detergent soluble Aβ ELISA from hippocampal tissues were also used to perform western blotting analysis. Proteins were separated by electrophoresis with the 12% SDS-PAGE and transferred onto PVDF membranes (Boster, China). The blot was probed with A8717 (Sigma, USA) to detect APP and CTFβ and rabbit anti-β-actin (Bioworld Technology, USA) to control for loading differences, followed by peroxidase-conjugated goat anti-rabbit IgG (Boster, China). The protein bands were visualized by ECL detection reagents (Applygen, China). Western blot images were quantitated using ImageJ (National Institutes of Health, USA). The ratios of target proteins over β-actin were calculated.
3
Adipose Tissue Protein Analysis
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Equal amount proteins extracted from adipose tissue or differentiated adipocytes of the mice were underwent SDS-PAGE and immunoblotting analysis with the primary antibodies and horseradish peroxidase-conjugated IgG. The blots were developed with enhanced chemiluminescence (ECL) detection reagents (Applygen Technologies, Beijing). Total RNA was extracted from adipose tissue and differentiating SVCs with use RNAtrip reagent (Applygen Technologies, Beijing) and reverse-transcribed to generate first-strand cDNA [33 (link)]. Real-time PCR was carried out in duplicate in an Mx3000 Quantitative PCR System (Stratagene). The relative target mRNA levels were analyzed and normalized to that of internal control 18S rRNA. The primer sets were listed in S1 Table .
4
Western Blot Analysis of Apoptotic and Glucose Metabolism Proteins
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Samples from pull-down assay were resolved in a 12% polyacrylamide gel and then transferred onto a PVDF membrane (Millipore, MA). After being blocked with 5% nonfat milk in TBS-T buffer (20 mM Tris, 137 mM sodium chloride at pH 7.6, 0.1% Tween-20) for 1 h at room temperature, membranes were incubated with primary antibody overnight at 4°C. Horse radish peroxidase (HRP)-conjugated (ZSGBBIO, Beijing, China) secondary antibodies were used to detect the immunoreactivity by enhanced chemiluminescence (ECL) detection reagents (Applygen Technologies, Beijing, China).
Western blotting analysis was also performed to determine the expression of certain apoptotic related or glucose-metabolism related proteins in cells treated with or without GRN A. Briefly, HepG-2 cells were seeded into 6 well plates at a density of 2 × 105 cell/well. After treated with or without GRN A for 48 h, the cells were collected and lysed with cold RIPA buffer (#R0278, Sigma-Aldrich, St. Louis, MO). The cell lysates were resolved on SDS-PAGE and analyzed using Western blotting experiments.
Western blotting analysis was also performed to determine the expression of certain apoptotic related or glucose-metabolism related proteins in cells treated with or without GRN A. Briefly, HepG-2 cells were seeded into 6 well plates at a density of 2 × 105 cell/well. After treated with or without GRN A for 48 h, the cells were collected and lysed with cold RIPA buffer (#R0278, Sigma-Aldrich, St. Louis, MO). The cell lysates were resolved on SDS-PAGE and analyzed using Western blotting experiments.
5
Western Blot Analysis of Protein Expression
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Whole cell lysates or immunoprecipitated samples were resolved in 10% SDS-PAGE gels and transferred to PVDF membrane (Millipore, MA). After being blocked with 5% non-fat dried milk for 1 hour at room temperature, membranes were incubated in primary antibody overnight at 4°C. Horse radish peroxidase (HRP)-conjugated (ZSGB-BIO, Beijing, China) or infrared fluorescent dyes (IRDye)-conjugated (LI-COR Biosciences, Lincoln, NE) secondary antibodies were used to detect the immunoreactivity by enhanced chemiluminescence (ECL) detection reagents (Applygen Technologies, Beijing, China) and Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE) respectively.
6
Western Blot Analysis of Autophagy Markers
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Heart tissues or cells were collected and lysed with RIPA buffer (Beyotime, P0013C) containing protease inhibitors (Selleck, B15002). The samples (20 µl) were used to determine the total protein concentrations with BCA reagent kit (Applygen, P1511-1). Protein samples were heat-denatured for 5 min at 95°C with the sample loading buffer, and 20 µg of total protein per sample were separated by SDS-PAGE gels and transferred to PVDF membranes (Millipore, IPVH00010). The membranes were blocked with 5% BSA for 30 min and incubated overnight at 4°C shaking with primary antibodies. The membranes were then washed in Tris-buffered saline (TBS, pH 7.6) with 0.1% Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibodies (1:4000, ZSGB-BIO, ZB2301) for 2 h at room temperature. Bands were visualized using ECL Detection Reagent (Applygen, P1050), and quantify analysis was performed using Gel-Pro Analyzer System. The primary antibodies used were presented as follow: anti-LC3B (1:5000, Sigma, L7543); anti-p62/SQSTM1 (1:5000, Sigma, P0067); anti-PINK1 (1:1000, Abcam, ab23707); anti-TOM20 (1:1000, Abcam, ab78547); anti-α-Actin (1:5000, Abcam, ab156302).
7
Western Blot Analysis of Protein Expression
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Protein lysates were prepared using RIPA lysis buffer (Thermo Fisher Scientific, Inc.). Protein concentration was determined using the Pierce bicinchoninic acid protein assay (Thermo Fisher Scientific, Inc.), and 50 µg protein/lane was separated on 4–15% Tris-glycine precast gels (Bio-Rad Laboratories, Inc.). Proteins were transferred onto a PVDF membranes and blocked with 5% non-fat milk at 25°C for 1 h, prior to incubation with primary antibodies against CREBBP (cat. no. sc-369; 1:500; Santa Cruz Biotechnology, Inc.), E2F3 (cat. no. sc-878; 1:500; Santa Cruz Biotechnology, Inc.), CASP8AP2 (cat. no. YT6423; 1:500; ImmunoWay Biotechnology, Inc.) or GAPDH (cat. no. 51332; 1:2,000; Cell Signaling Technology, Inc.) overnight at 4°C. Membranes were washed three times for 10 min with TBST buffer (cat. no. B1009; Applygen Technologies, Inc.) and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or anti-mouse secondary antibody (cat. nos. 7074 and 7076, 1:5,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. Blots were re-washed and signals were visualized with super enhanced chemiluminescence (ECL) detection reagent (Applygen Technologies, Inc.), then imaged using Amersham Imager 600 (Cytiva) and analyzed with ImageQuant™ TL software (version 7.0; Cytiva).
8
Western Blot Analysis of Cell Signaling
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Cell lysates were extracted from cells using RIPA lysis buffer. Proteins were separated by SDS–PAGE (10% polyacrylamide), transferred to a PVDF membrane (Millipore), and blocked in 5% dry milk in Tris-buffered saline with Tween 20 (TBST; Applygen Technologies, Beijing, China). Membranes were incubated with primary antibody, and target proteins were detected with enhanced chemiluminescence (ECL) using ECL detection reagent (Applygen Technologies, Beijing, China). Primary antibodies included the following: AKT (Cell Signaling Technology Cat# 9272), phospho-AKT (Cell Signaling Technology Cat# 4060), AMPKα (D5A2) (Cell Signaling Technology Cat# 5831), phospho-AMPKα (Thr172) (40H9) (Cell Signaling Technology Cat# 2535), mTOR (Cell Signaling Technology Cat# 2983), phospho-mTOR (Abcam Cat# ab109268), p70S6K (Proteintech Cat# 14485-1-AP), phospho-p70S6K (Cell Signaling Technology Cat# 9234), 4eBP1 (Proteintech Cat# 60246-1-Ig), phospho-4eBP1 (Cell Signaling Technology Cat# 2855), GAPDH (Proteintech Cat# 60004-1-Ig), CDK1 (Santa Cruz Biotechnology Cat# sc53219), CDK4 (Cell Signaling Technology Cat# 12790), CDK6 (Abcam Cat# ab124821), Rb (Cell Signaling Technology Cat# 9309), and phospho-Rb (Ser795) (Cell Signaling Technology Cat# 9301).
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