To evaluate the expression and distribution of LC3-I, LC3-II, beclin-1, mTOR and PI3K protein, total protein was extracted using radioimmunoprecipitation assay buffer, following which the protein concentration was determined using a BCA Protein assay kit (cat. no. 23225; Bio-Rad Laboratories Inc., Hercules, CA, USA). Proteins were added to protein sample buffer, boiled in water for 5 min and separated by SDS-PAGE. Proteins were transferred onto polyvinylidene fluoride membranes and Blocked at 4°C overnight with Tris-buffered saline/Tween 20 (TBST) containing 5% non-fat dried milk. Membranes were incubated with anti-beclin1 (1:1,000), anti-LC3 (1:1,000), anti-PI3K (1:1,000), anti-mTOR (1:1,000) and anti-GAPDH antibodies (1:2,500; cat. no. sc-365062; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) primary antibodies, at 4°C overnight. The membranes were rinsed three times with PBS Tween20 and then incubated with the secondary antibodies (1:2,500) for 2 h at room temperature. The protein bands were visualized using an enhanced chemiluminescent kit (EMD Millipore, Billerica, MA, USA). ImageJ software (Version 4.0; National Institutes of Health, Bethesda, MD, USA) was used to analyze the grayscale values of each group and GAPDH was used as the internal reference.
Enhanced chemiluminescent kit
Manufactured by Merck Group
Sourced in United States
The Enhanced chemiluminescent kit is a laboratory product developed by Merck Group. It is used to detect and quantify proteins in western blot analysis. The kit contains reagents that generate a chemiluminescent signal when in contact with the target protein, allowing for its visualization and measurement.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (6 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Sds page, by Merck Group (2 mentions)
Chemiluminescence detection system, by Bio-Rad (1 mentions)
Total protein extraction kit, by Merck Group (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Gel pro analyzer densitometry software, by Media Cybernetics (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Loading buffer, by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Beyotime (1 mentions)
Bicinchoninic acid protein assay kit, by Merck Group (1 mentions)
Anti gapdh, by BD (1 mentions)
Anti snail, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
18 protocols using enhanced chemiluminescent kit
1
Western Blot Analysis of Autophagy Proteins
2
Quantitative Analysis of Wnt and β-catenin Proteins in hMSCs
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Total protein was extracted from hMSCs using the Total Protein Extraction kit (Sigma) according to the manufacturer’s protocols. The protein concentration was determined with a BCA Protein Assay kit (Thermo scientific, Rockford, Illinois, USA) according to the manufacturer’s instructions. Then 40 μg protein were separated by SDS-PAGE, and then were transferred onto polyvinylidene difluoride membrane. The membrane was incubated with mouse polyclonal antibodies specific for Wnt 11 (1:150; Santa Cruz, CA, USA) and β-catenin (1:1000; Santa Cruz) overnight at 4 °C. After washing with Tris-buffered saline containing 0.1 % Tween, the membranes were incubated with secondary antibody (1:5000; Santa Cruz) for 1 hour. Finally, the membrane was exposed, visualized using an enhanced chemiluminescent kit (Merck Millipore, Eschborn, Germany) and a chemiluminescence detection system (Bio-Rad, Hercules,California, USA). Protein bands were quantified using the Quantity One software (Bio-Rad).
3
Apoptotic Pathway Protein Analysis
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Cells were lysed with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) following MET and PTX treatment for 24 h. Total protein was extracted at 4°C and concentration was determined using a bicinchoninic acid assay. Proteins (30 µg protein) were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA) at 4°C. The membrane was blocked in 5% milk in Tris-buffered saline with 1% Tween-20 at room temperature for 1 h, and incubated with primary antibodies overnight at 4°C at the following dilutions: PARP, 1:1,000; caspase-3, 1:1,000; caspase-9, 1:1,000; Bcl-2, 1:1,000; Bax, 1:1,000; Cyto-C, 1:1,000; P53, 1:1,000; and GAPDH, 1:100,000. The membranes were then probed with IgG-HRP antibody (dilution, 1:10,000) for 1 h at room temperature. Finally, the proteins were detected using Enhanced Chemiluminescent kit (EMD Millipore).
4
Western Blot Analysis of Protein Expression
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Protein expression was measured by western blotting, as described previously (3 (link),6 (link)), with some modifications. The precipitate protein was lysed in lysis buffer (Beyotime Institute of Biotechnology), and protein concentrations were measured using a Bradford protein assay kit (Bio-Rad Laboratories, Inc.). Equal amounts of protein (15 µg) were separated via SDS-PAGE with an 8% gel and then transferred onto polyvinylidene difluoride membranes. Membranes were subsequently blocked with 5% nonfat milk in TBS with 0.1% Tween-20 (TBS-T) at room temperature for 1.5 h to eliminate non-specific binding. Membranes were then incubated overnight with primary antibodies against Sp1 (1:1,000; cat. no. ab124804; Abcam) and GADPH (1:3,000; cat. no. 60004-1-Ig; ProteinTech Group, Inc.) at 4°C. Subsequently, membranes were washed three times in TBS-T and incubated with secondary antibodies 2 h at room temperature (horseradish peroxidase-conjugated anti-rabbit immunoglobulin G; 1:3,000; cat. nos. WB0177 and A23210; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.). Immunoreactive bands were then visualized using an enhanced chemiluminescent kit (EMD Millipore) according to the manufacturer's instructions. Band densities were quantified using Gel-Pro Analyzer densitometry software (version 6.3; Media Cybernetics, Inc.
5
Western Blot Analysis of Testicular Proteins
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After sacrifice, approximately 60 mg of the testes from the circadian desynchrony and control mice of ZT0 batch was ground into powder and lysed in RIPA buffer (Beyotime, China) on ice. Total protein content was estimated using the BCA Protein Assay Kit (Beyotime, China), and lysates were mixed with 5 × loading buffer (Beyotime, China), heated at 100°C for 5 min, fractionated on 6–15% SDS-PAGE gels, and transferred to polyvinylidene difluoride membranes (Merck, USA). Membranes were blocked for 1 h with 5% bovine serum albumin in Tris-buffered saline containing 0.1% (v/v)Tween-20 (TBST; pH 7.4), incubated with primary antibodies at 4°C for 12 h, washed in TBST, and finally incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; Beyotime) for 1 h at room temperature. Immunoreactive bands were visualized using an enhanced chemiluminescent kit (Merck, USA). Protein levels were normalized to those of β-actin and expressed as a percentage of the levels in control animals.
6
Protein Expression Analysis in MeT-5A Cells
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MeT-5A cells were harvested and lysed using RIPA lysis buffer (EMD Millipore) for 15 min on ice. The total proteins were isolated by centrifugation at 12,000 ×g for 15 min at 4℃, and quantified using a bicinchoninic acid protein assay kit (EMD Millipore). Thirty µg total proteins were separated on 8-10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were subsequently blocked in 5% non-fat milk and then incubated with the corresponding primary antibodies, including Rabbit anti-Claudin-1 (1:1,000; cat. No. 13255, Cell Signaling Technology), anti-Snail (1:1,000; cat. No. 3879, Cell Signaling Technology), anti-Slug (1:1,000; cat. No. 9585, Cell Signaling Technology), anti-Vimentin (1: 1,000, cat. No. 550513, BD Biosciences), anti-GAPDH (1:5,000; cat. No. MAB374, EMD Millipore) antibodies at 4℃ overnight. The membranes were probed with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies, and the signals of detected proteins were visualized using an enhanced chemiluminescent kit (EMD Millipore).
7
Western Blot Quantification of MUC5AC
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Protein concentrations were measured by using the bicinchoninic acid protein assay. Protein samples were separated on 8% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, USA). Non-specific binding to the membrane was blocked for 1 h at room temperature with 5% fat-free milk in TBST, and then the membranes were incubated with 1 : 400 MUC5AC primary antibody (Santa Cruz, USA) at 4°C overnight. Then, the membrane was washed 4 times with TBST and then incubated with a 1 : 5000 dilution of the appropriate secondary antibody at room temperature for 45 min. After the membrane was washed twice with TBST, membrane-bound antibody was visualized by using an enhanced chemiluminescent kit (Millipore) according to the manufacturer’s instructions.
8
Protein Quantification and Western Blot
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Samples were lysed in RIPA lysis buffer. The bicinchoninic acid assay (BCA assay) was performed to quantify protein concentrations. Equal amounts of protein were separated by SDS-PAGE using 10% polyacrylamide. Next, proteins were transferred to PVDF membranes. Membranes were blocked with 5% fat-free milk, cut into strips, and incubated with primary antibodies and secondary antibodies. Detection was performed using an enhanced chemiluminescent kit (Millipore, MA, USA). Images were acquired using a ChemiDoc XRS system with Quantity One software (Bio-Rad, Richmond, CA, USA).
9
Comprehensive Protein Expression Analysis
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Total protein was extracted from cells treated with EVO or DMSO by using the mammalian protein extraction reagent RIPA (Beyotime, China). The protein concentration was measured using BCA (bicinchoninic acid) protein assays (Beyotime, China). Protein samples were boiled with 6× SDS loading buffer for 5 min, separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Membranes were blocked in 5% bovine serum albumin (BSA; Solarbio, Beijing, China) for 2h and then incubated with diluted specific primary antibodies overnight at 4°C. β-Actin was purchased from Zoonbio Technology (Beijing, China), while PCNA, β-catenin, c-Myc, cyclin D1, GSK-3β, Bcl-2, Bad, Bax, Caspase-3, cleaved Caspase-3, PARP, cleaved-PARP, MMP-2, MMP-7, MMP-9, vimentin, E-cadherin, N-cadherin and Snail were purchased from Cell Signaling Technology (Danvers, CO, USA). Primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies. The bands were visualized using an enhanced chemiluminescent kit (Millipore, USA) according to the manufacturer’s protocol. The protein bands were pictured and analyzed by using the ChemiDoc MP Imaging System and Image Lab Software (Bio-Rad, California, USA).
10
Western Blot Analysis of Protein Signaling
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Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). Then, we separated the equal amount of sample proteins (40 μg) on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred them onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) which contained 5% BSA for 1 hour and incubated with specific primary antibodies, including anti-extracellular signal-regulated kinase 1/2 (ERK1/2) (1:1000; CST, Danvers, MA), anti-p-ERK1/2 (1:1000; CST), anti-signal transducer and activator of transcription 3 (STAT3) (1:1000; CST), anti-p-STAT3 (1:2000; CST), and anti-β-actin (1:1000; CST) overnight at 4°C. The membranes were washed three times in TBST and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:10000) for 1 hour at room temperature. Protein bands were visualized by an enhanced chemiluminescent kit (Millipore, Bedford, MA) and densitometric analysis was performed using Image J 1.47. The β-actin served as an internal control.
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