Cells were harvested and lysed in immune precipitation assay buffer (KeyGen Biotech Co., Ltd, Nanjing, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (KeyGen Biotech Co., Ltd, Nanjing, China) and 1 mM phosphatase inhibitor cocktail (KeyGen Biotech Co., Ltd) for extracting whole cell protein samples. Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (KeyGen Biotech Co., Ltd). Equal amounts of protein samples were separated on 10% SDS-PAGE gels and then transferred onto polyvinylidene difluoride membranes (Pall Corporation, Port Washington, NY, USA). The protein band intensities were evaluated using an electrochemiluminescence (ECL) western blotting kit (Advansta, Menlo Park, CA, USA) and were normalized to GAPDH or β-tubulin. All experiments were performed at least three times.
Ecl western blotting kit
Manufactured by Advansta
Sourced in United States
The ECL western blotting kit is a set of reagents designed for the detection and visualization of proteins in western blot analysis. The kit includes solutions for blocking, washing, and developing the blot, as well as the necessary chemiluminescent substrate for generating a signal.
Automatically generated - may contain errors
Lab products found in correlation
Immune precipitation assay buffer, by Keygen Biotech (2 mentions)
Phosphatase inhibitor cocktail, by Keygen Biotech (2 mentions)
Bca protein assay kit, by Keygen Biotech (2 mentions)
Polyvinylidene difluoride membrane, by Pall Corporation (2 mentions)
Phenylmethylsulfonyl fluoride, by Keygen Biotech (1 mentions)
Ab6319, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Ab1791, by Abcam (1 mentions)
Sc 9996, by Merck Group (1 mentions)
Axio imager z2 microscope system, by Zeiss (1 mentions)
5 protocols using ecl western blotting kit
1
Whole Cell Protein Extraction and Western Blot
2
Quantitative Protein Analysis of Brain Tissues
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The hippocampus and cerebral cortex tissues were homogenized and centrifuged, and supernatants were extracted. All of the samples were quantitatively analyzed using BCA for total protein content. The 25 ug of protein was run on a 10% gradient SDS–PAGE gel and electrophoretically transferred to polyvinyl difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Nonspecific binding proteins were blocked in 5% milk for 1.5 h at room temperature. Incubated protein bands with primary antibodies were left overnight at 4 °C before the membranes were incubated with antimouse IgG or antirabbit IgG antibodies for 2 h at room temperature; the antibodies were horseradish-peroxidase-labeled. Blots were visualized using an ECL western blotting kit (Advansta). Protein bands were scanned and semiquantified with densitometry using ImageJ software. The primary antibodies used were MBP (10458-1-AP, Proteintech, Wuhan, China), CNpase (ab6319, Abcam, Cambridge, UK), MAG (DF7669, Affinity, Liyang, China), and MOG (12690-1-AP, Proteintech, Wuhan, China).
3
Western Blot Analysis of NOX4 Expression
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Total protein from MPC-5 was extracted with RIPA lysis buffer. After HG stimulation for different time points, cell lysis samples were subjected to SDS/PAGE electrophoresis, followed by transfer to PVDF membranes, and blockage for 2 h at room temperature with 5% skim milk. The primary antibody rabbit polyclonal anti-NOX 4 (1:2000) or Mouse monoclonal anti-β-Tubulin (1:5000) was incubated overnight at 4°C on the shaker. The membranes were incubated with horseradish peroxidase–conjugated goat anti-rabbit or anti-mouse antibody (Sungene Biotech, Tianjin, China). The specific proteins were detected using an enhanced chemiluminescence (ECL) Western blotting kit (Advansta, San Jose, CA). ImageJ was applied for targeted bands analysis.
4
Protein Extraction and Localization Analysis
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Cells were harvested and lysed in immune precipitation assay buffer (KeyGen Biotech Co., Ltd, Nanjing, China) supplemented with 1 mM phenylmethylsulfonyl floride (KeyGen Biotech Co., Ltd, Nanjing, China) and 1 mM phosphatase inhibitor cocktail (KeyGen Biotech Co., Ltd) for extracting whole cell protein samples. For detecting the cellular localization of total MDM2 and phos-MDM2, nuclear and cytoplasmic fractions were isolated using the Nuclear and Cytoplasmic Protein Extraction kit (Thermo Fisher Scientific) according to the instructions of the manufacturer. Protein concentration was determined using a BCA protein assay kit (KeyGen Biotech Co., Ltd). Same amount protein samples were separated on 10% SDS-PAGE gels and then transferred to a polyvinylidene difluoride membrane (Pall Corporation, Port Washington, NY, USA). The protein band intensities were evaluated using ECL western blotting kit (Advansta, Menlo Park, CA, USA) and were normalized to those of β-actin. All experiments were performed at least three times.
5
Visualizing and Quantifying WRKY63 Expression
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The vernalization or non-vernalization treated WRKY63pro::WRKY63:GFP transgenic plants were used for the fluorescence imaging. GFP fluorescence was visualized by using the Zeiss Axio Imager.Z2 microscope system (https://www.zeiss.com/microscopy). The GFP signal was quantified in the middle section of cotyledons for analyzing GFP intensity by the Zeiss ZEN software (https://www.zeiss.com/microscopy/int/products/microscope-software/zen.html). GFP fluorescence was observed through the filter sets 46 HE (excitation at 500 nm, beamsplitter at 515 nm, and emission at 535 nm). Images were captured using a 5× objective with tile scan mode. The vernalization and non-vernalization samples were captured in the same field together and then processed and analyzed using the Zeiss ZEN software as described above.
Western blot assays were performed as previously described (Hung et al., 2018) . Anti-GFP (Santa Cruz Biotechnologies, catalog no. SC-9996; 1:3,000 dilution), anti-FLAG (sigma, M2; 1:3,000 dilution), and anti-H3 (Abcam, ab1791; 1:3,000 dilution) antibodies were used as primary antibodies for Western blot; the resulting signals were detected by using an Advansta ECL Western blotting kit (Advansta, K-12045-D20).
Western blot assays were performed as previously described (Hung et al., 2018) . Anti-GFP (Santa Cruz Biotechnologies, catalog no. SC-9996; 1:3,000 dilution), anti-FLAG (sigma, M2; 1:3,000 dilution), and anti-H3 (Abcam, ab1791; 1:3,000 dilution) antibodies were used as primary antibodies for Western blot; the resulting signals were detected by using an Advansta ECL Western blotting kit (Advansta, K-12045-D20).
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