Total RNA was extracted from cells using TRIzol™ LS Reagent (Thermo Fisher Scientific #10296010) or RNeasy Plus Mini Kit (Qiagen #74134). mRNA was isolated and purified using the Dynabeads™ mRNA purification kit (for mRNA purification from total RNA preperations; Invitrogen #61006) following the manufacturer's instructions. For the m6A dot blot, mRNA was hybridized onto the Hybond-N+ membrane (GE Healthcare). After crosslinking spotted mRNA to the membrane using a Stratalinker 2400 UV Crosslinker, the membrane was blocked with 5% skimmed milk for 1 h, and incubated with mouse anti-m6A antibody (1:1000, Millipore #MABE1006) at 4°C overnight. Then the membrane was incubated with horseradish peroxidase (HRP)-conjugated donkey anti-mouse-IgG at room temperature for 1 h. The membrane was photographed using the ECL imaging system (Bio-Rad). Finally, the membrane was stained with 0.02% Methylene Blue. The relative m6A level was quantified using ImageJ.
Ecl imaging system
Manufactured by Bio-Rad
Sourced in United States
The ECL imaging system is a laboratory instrument designed for the detection and quantification of chemiluminescent signals in Western blot analysis. It provides a sensitive and reliable method for visualizing and analyzing protein expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon western chemiluminescent hrp substrate, by Merck Group (5 mentions)
Hybond n membrane, by GE Healthcare (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ibfp0785c, by Bio-Rad (3 mentions)
Semi dry transfer instrument, by Bio-Rad (3 mentions)
Electrophoresis apparatus, by Bio-Rad (3 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (2 mentions)
Mabe1006, by Merck Group (2 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Rabbit anti m6a antibody, by Synaptic Systems (2 mentions)
Non fat milk, by Bio-Rad (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bca assay kit, by Beyotime (1 mentions)
Polyattract mrna isolation system 3, by Promega (1 mentions)
Bca protein assay kit, by Takara Bio (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Protein quantification kit, by Transgene (1 mentions)
Anti cith3, by Abcam (1 mentions)
22 protocols using ecl imaging system
1
Quantifying m6A Methylation in mRNA
2
Western Blot Analysis of Mouse Pancreatic Islets
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Protein was extracted from the mouse pancreatic islet by using RIPA lysis buffer and measured by a BCA assay kit (Beyotime, Jiangsu, China). A total of 30 μg protein was separated by 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Then, the membranes were blocked with nonfat milk and incubated with primary antibodies at 4°C overnight. HRP‐conjugated IgG secondary antibodies were used to combine with the primary antibodies and visualized by an ECL imaging system (Bio‐Rad, California, USA).
3
Western Blot Analysis of Protein Expression
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Cells were lysed with RIPA buffer containing 1% PMSF followed by ultrasonication. Cell lysates were incubated on ice for 30 min, centrifuged at 10,000 × g for 10 min. The supernatants were collected and the protein concentration was detected using a BCA detection Kit. Equal amount of proteins was loaded to the polyacrylamide gel. The proteins were separated through SDS-PAGE using the electrophoresis apparatus (Bio-Rad). After electrophoresis, the proteins were transferred to the PVDF membrane (Millipore, IBFP0785C) using a semi-dry transfer instrument (Bio-Rad). The membranes were blocked with 5% non-fat milk for 1 h at room temperature, incubated with primary antibodies at 4 °C overnight. Subsequently, the membranes were washed with PBST and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. After washing, the membranes were incubated with the Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA) and photographed using the ECL imaging system (Bio-Rad, USA).
4
m6A RNA methylation quantification
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Total RNA was extracted from the cells using Trizol reagent (TAKARA). mRNA was isolated and purified using Poly Attract mRNA Isolation System III with Magenetic Stand (Promega) following the manufacturer’s instructions. For m6A dot blot, mRNA was hybridized onto the Hybond-N + membrane (GE Healthcare). After crosslinking at 80 °C for 30 min, the membrane was blocked with 5% non-fat milk (Biorad) for 1 h, incubated with rabbit anti-m6A antibody (1:1000, Synaptic Systems, cat. No. 202003) at 4 °C overnight. Then the membrane was incubated with HRP-conjugated mouse anti-rabbit IgG (1:3000, Santa,sc-2357) at room temperature for 2 h. After being incubated with Immobilon Western Chemiluminescent HRP Substrate (Millipore), the immunocomplex was photographed using the ECL imaging system (Bio-Rad). Finally, the membrane was stained with 0.02% methylene blue to eliminate the difference in mRNA amount. Relative m6A level was quantified via gray intensity analysis using Image J.
5
Western Blot Protein Detection Protocol
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Cells were lysed with RIPA lysis buffer (Solarbio Life Sciences, R0020) containing 1% PMSF followed by ultrasonication. Cell lysates were incubated on ice for 30 min, followed by centrifuging at 4 °C for 10 min. The protein concentration of the supernatant was quantified using a BCA Protein Assay Kit (TAKARA, T9300A). The proteins were separated through SDS-PAGE using the electrophoresis apparatus (Bio-Rad). Subsequently, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, IBFP0785C) using a semi dry transfer instrument (Bio-Rad). The membranes were blocked with 5% non-fat milk at room temperature for 1 h, and incubated with primary antibodies at 4 °C overnight. After overnight incubation, the membranes were washed with PBST and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h, followed by incubation with the Immobilon Western Chemiluminescent HRP Substrate (Millipore) and photographed using the ECL imaging system (Bio-Rad).
6
Western Blot Analysis of Inflammatory Markers
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Protein was extracted from lysis buffer containing a protease inhibitor, and the level of protein was quantified with a protein quantification kit (TransGen Biotech, Beijing, China). Then, equivalent protein samples were separated by SDS-PAGE gels and transferred onto PVDF membranes (Merck Millipore, Darmstadt, Germany), which were blocked in Protein-free fast blocking solution (Boster, China) and cultivated with primary antibody for 12h at 4 ℃. After washing three times with wash buffer, the membrane was incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at 37C°. Protein bands were intuitively visualized by an enhanced chemiluminescence (ECL) imaging system (Bio-Rad Laboratories, CA, USA) and quantified with ImageJ software (Rawak Software Inc., Germany). Principal primary antibodies were as follows: rabbit polyclonal anti-CitH3 (1:1000, Abcam, UK), rabbit polyclonal anti-NLRP3 (1:1000, Abcam, UK), rabbit polyclonal anti-GSDMD (1;1000, Cell single Technology, USA), rabbit polyclonal anti-caspase-1 (1:1000, Abcam, UK), rabbit polyclonal anti-H3 (1:1000 dilution, Immunoway Biotechnology, USA), rabbit polyclonal anti-caspase-1 p20 (1:500, Novus Biologicals LLC, USA), and rabbit polyclonal anti-GAPDH (1:1000, Proteintech, China).
7
Western Blot Analysis of p-p38 MAPK
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The protein concentration of the fresh lung tissue was measured using the Bradford method; equal amounts of protein were separated by electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, MA). Nonspecific antibody binding was blocked by incubating the membrane with 5% bovine serum albumin in Tris-buffered saline with Tween-20 (TBST) for 1 h. Subsequently, the membrane was incubated overnight at 4°C with specific rabbit anti-rat p-p38 MAPK polyclonal (1 : 1000) and rabbit anti-rat GAPDH antibodies (1 : 1000). After it was washed with fresh TBST, the membrane was incubated with HRP-conjugated, affinity-purified anti-rabbit IgG (0.1 μg/mL). Immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) kit (Pierce, USA) and an ECL Imaging System (Bio-Rad, USA). The signal intensities of the corresponding bands were quantified using Image-Pro Plus software.
8
Protein Expression Analysis in Prostate and Lung Cancer Cells
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LNCaP and NCI-H660 cells were harvested using radioimmunoprecipitation assay (RIPA) Lysis (Elabscience, Wuhan, China), and protein concentration were detected by the BCA protein assay kit (Pierce, Rockford, IL, USA). 10 µg of protein were separated by 10% SDS-PAGE gels, followed by electrotransfer onto PVDF membranes (Arkema, Paris, France). The membrane was incubated with primary antibody against ChgA (1:1000, ab254557, Abcam), NSE (1:100, ab105389, Abcam), SYP (1:1000, ab32127, Abcam), Hnrnpa2b1 (5 µg/ml, ab31645, Abcam), and β-actin (1:1000, 3700, CST, Danvers, MA, USA) overnight at 4 °C, HRP-conjugated secondary antibodies (1:1000; ab205718; Abcam), and visualized through the ECL imaging system (Bio-Rad, Hercules, CA, USA).
9
Immunoprecipitation and Western Blot Analysis
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HEK-293T cells were transfected with the indicated plasmids as mentioned above, harvested after 30 h by the addition of lysis buffer, and incubated on ice for 15 min. The cell lysates were centrifuged at 12,000 × g for 15 min before the supernatants were either subjected to immunoprecipitation (IP) or directly denatured at 100°C for 10 min. The denatured cell lysates were separated by SDS−PAGE and transferred to PVDF membranes using a Trans-Blot® SD Semi-Dry Electrophoretic Transfer cell at 15 V for 50 min. For immunoblotting, the indicated primary antibodies were incubated with the membranes for 2 h at room temperature or overnight at 4°C; HRP-conjugated goat anti-mouse or goat anti-rabbit IgG were used as secondary antibodies. The bands were visualized with chemiluminescent reagent (NCM Biotech, Shanghai, China) and were imaged by an ECL imaging system (Bio-Rad, Hercules, CA, USA). GAPDH expression in each sample was used as a control to demonstrate equal loading of the protein samples across lanes.
10
Western Blot Analysis of SHC3
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Cells were harvested 72 h after transduction, washed twice with PBS, and homogenized in lysis buffer on ice. The protein concentration in the lysates was measured using the BCA reagent. Following SDS-PAGE, nitrocellulose membrane transfer and blocking, the blots were incubated overnight at 4 °C with anti-SHC3 (1:500; Sigma, SAB1401715) and anti-GAPDH (1:10,000; Abcam, ab37168) antibodies. The blots were then incubated with the secondary antibodies at room temperature for 1 h, and the protein bands were detected by enhanced chemiluminescence (ECL) reagents (Millipore; Billerica, MA, USA) and ECL imaging system (Bio-Rad; Richmond, CA, USA) according to the manufacturer’s instructions.
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