Cells were lysed in polysome lysis buffer (100 mM KCl, 5 mM MgCl2, 20 mM Tris, pH 7.4, 0.5% NP-40) supplemented with HALT protease and phosphatase inhibitor mixture (Roche). Lysates were centrifuged at 15 000 × g for 10 min and supernatants collected. Protein concentration was determined using the Pierce 660-nm protein assay (Thermo Scientific). Proteins were separated by SDS-PAGE, transferred onto PVDF and blocked with 5% (v/v) non-fat milk in 0.1% (v/v) Tween-20 in TBS. Membranes were incubated with primary antibodies against XIAP (610762, 1:2000), SOD2 (611580, 1:1000; BD Biosciences), p-NFκB (3032, 1:1000), NFκB (8242, 1:1000), SOD1 (2770, 1:1000; Cell Signaling) and GAPDH (47724, 1:4000; Santa Cruz Biotechnology), overnight at 4 °C. Immunoreactive bands were detected using horseradish peroxidase-conjugated secondary antibodies (Cell Signaling) in combination with chemiluminescence ECL (Thermo Scientific). Stripping of membranes for detection of total protein was performed as described previously.60 (link) Densitometric analysis was conducted using the NIH ImageJ software.63
Ecl chemiluminescence
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
ECL chemiluminescence is a laboratory technique used to detect and quantify specific proteins or molecules in a sample. It utilizes the principle of chemiluminescence, where a luminescent reaction occurs when a chemical substrate interacts with an enzyme, resulting in the emission of light. This technique is commonly used in Western blotting and other immunoassays to visualize and analyze target proteins.
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Lab products found in correlation
Pvdf membrane, by Merck Group (11 mentions)
Protease inhibitor cocktail, by Roche (6 mentions)
Ripa buffer, by Beyotime (4 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Nitrocellulose, by Bio-Rad (3 mentions)
Bp152 5, by Thermo Fisher Scientific (3 mentions)
Bp120 1, by Merck Group (3 mentions)
E3889, by Merck Group (3 mentions)
S6508, by Roche (3 mentions)
L3771, by IBI Scientific (3 mentions)
Ib74040, by Merck Group (3 mentions)
β actin, by Merck Group (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
Nupage novex bis tris gel system, by Thermo Fisher Scientific (3 mentions)
Dithiothreitol (dtt), by Avantor (2 mentions)
G5516, by Merck Group (2 mentions)
Villin, by Santa Cruz Biotechnology (2 mentions)
Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
47 protocols using ecl chemiluminescence
1
Western Blot Analysis of Cellular Proteins
2
TRPA1 Signaling Pathway Regulation
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TRPA1 (19124-1-AP) was purchased from Proteintech Group (Rosemont, IL 60018, USA). CaMKII (4436), pCaMKII (12716), β-catenin (8480) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). Retinal (R2500), JT010 (SML1672), HC030031 (H4415) were purchased from Sigma Aldrich (Darmstadt, Germany). Dimethyl sulfoxide (DMSO), RIPA buffer (R0100), BCA protein assay kit (PC0020), poloxamer gel (Polyethylene-polypropylene glycol 407, S7071) and formalin (G2161) were purchased from Solarbio (Beijing, China). Protease inhibitor cocktail (B14011) was purchased from Bimake (Shanghai, China). XAV-939 (S1180) was purchased from Selleck Chemicals (Shanghai, China). Fluo-4 AM (ab142773), DIO Staining Solution (C1038) were purchased from Beyotime (Shanghai, China). Stock solutions were prepared as follows: HC030031, and XAV-939 were dissolved in DMSO, JT010, and retinal in absolute ethanol. Masson-Fontana staining solution (G2032) was purchased from Solarbio (Beijing, China). Opti-MEM medium (31985062), Lipofectamine 2000 (11668030) and chemiluminescence (ECL) were obtained from Thermo Fisher Scientific (Massachusetts, USA). The UVA or UVB source used was a 9W UVA or UVB broadband lamp (Philips, Eindhoven, Netherlands) and radiation energies were measured using a UVX radiometer (UVP, Upland, California, USA).
3
Isolation and Analysis of Mouse Ileal Epithelial Cells
4
Smad1/5/8 Phosphorylation Assay
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Cells were treated according to the experiment conditions and then collected at specific time points, washed in cold PBS buffer and lysed in cell lysis buffer (10 mM Tris–HCl, pH 7.4, 150 mM sodium chloride, 1% NP-40, 0.1% SDS, 1 mM EDTA, 1 mM phenyl-methylsulfonyl fluoride, 1 mM NaF, 1 mM Na3VO4), supplemented with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). 20 μg of protein was separated on 8–12% NuPAGE® Novex® Bis-Tris gel systems (Thermo Fisher Scientific, Germany) followed by transfer to PVDF membrane (Millipore, USA). The membrane was blocked and incubated with peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Heidelberg Germany). Proteins were visualized by ECL chemiluminescence (Thermo Fisher Scientific, Roskilde, Germany). Antibodies for Smad1/5/8 (total or phosphor) were obtained from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Quantification of western blots was performed with ImageJ program.
5
Western Blot Analysis of Epigenetic Regulators
6
Western Blot Analysis of PTEN-Akt Signaling
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The cells (NCI-H1975 or A549 at 80% confluence in 6-well plates) were lysed with RIPA buffer (Thermo Fisher Scientific, Inc.) and the protein concentration was determined using the bicinchoninic acid kit (Beyotime Institute of Biotechnology). Proteins (20 µg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (MilliporeSigma). Following blocking (with 5% nonfat milk in TBST at room temperature for 1 h), the membrane was incubated overnight at 4°C with the primary antibodies: PTEN (1:1,000; cat. no. ab267787), p-Akt (1:500; cat. no. ab131443), Akt (1:10,000; cat. no. ab179463), p-PI3K (1:500; cat. no. ab182651), PI3K (1:1,000; cat. no. ab191606) and GAPDH (1:1,000; cat. no. ab9485; all Abcam). Subsequently, PBST was used to rinse the membranes three times. Secondary antibody goat anti-rabbit IgG H&L (HRP; cat. no. ab6721; Abcam) was used to incubate the membranes at room temperature for 1 h. All protein bands were visualized using ECL chemiluminescence (Thermo Fisher Scientific, Inc.). Protein bands were visualized in a gel imaging system (MG8600, Bio-Rad). Images were using Image Pro Plus software (Version 7.0, Media Cybernetics).
7
Immunoblot Detection of ANGPTL4 on HDL Fractions
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HDL fractions (10 μL) were separated using 10% SDS‐PAGE. After transfer, nitrocellulose membranes were probed with anti‐ANGPTL4 antibody 1/1000 (Abcam, Cambridge, UK) overnight and then incubated with horseradish peroxidase–conjugated IgG secondary antibody 1/3000 (Amersham Biosciences, Piscataway, NJ) for 1 hour. Signals were detected by ECL chemiluminescence (Thermo Fisher Scientific, Rockford, IL).
To further confirm the presence of ANGPTL4 on HDL fractions, HDLs (400 μL) and lysis buffer (400 μL) were incubated with anti‐ANGPTL4 antibody (Santa Cruz, Dallas, TX) or IgG (Cell Signaling Technology, Beverly, MA) coupled with protein G plus/protein A‐agarose (Santa Cruz, Dallas, TX) overnight. After washing, the immunoprecipitated proteins were processed for Western blotting as described above.
HEK 293 cells transfected with plasmid containing EL cDNA (GFP‐EL‐flag) were lysed in 1 mL ice‐cold lysis buffer (10 mmol/L HEPES, 50 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L benzamidine, and 0.5% Triton X‐100 [pH 7.4]). The lysates were analyzed by Western blotting with anti‐flag antibody (Medical and biological laboratories, Nagoya, Japan) and anti‐GAPDH antibody (Santa Cruz, Dallas, TX).
To further confirm the presence of ANGPTL4 on HDL fractions, HDLs (400 μL) and lysis buffer (400 μL) were incubated with anti‐ANGPTL4 antibody (Santa Cruz, Dallas, TX) or IgG (Cell Signaling Technology, Beverly, MA) coupled with protein G plus/protein A‐agarose (Santa Cruz, Dallas, TX) overnight. After washing, the immunoprecipitated proteins were processed for Western blotting as described above.
HEK 293 cells transfected with plasmid containing EL cDNA (GFP‐EL‐flag) were lysed in 1 mL ice‐cold lysis buffer (10 mmol/L HEPES, 50 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L benzamidine, and 0.5% Triton X‐100 [pH 7.4]). The lysates were analyzed by Western blotting with anti‐flag antibody (Medical and biological laboratories, Nagoya, Japan) and anti‐GAPDH antibody (Santa Cruz, Dallas, TX).
8
Western Blot Analysis of Colonic Smooth Muscle
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Colonic smooth muscle tissues and cultured colonic SMCs were lysed in RIPA buffer (Applygen, Beijing, China) with 1% phosphatase inhibitor cocktail (Sigma‐Aldrich), 1% protease inhibitor cocktail (Sigma‐Aldrich) and 1% phenylmethanesulphonyl fluoride (PMSF, Solarbio, Beijing, China). Proteins were separated on 10% SDS‐PAGE gel and transferred onto PVDF membrane (Merk‐Millipore, Temecula, CA, USA), followed by blockage with 5% non‐fat milk or bovine serum albumin (BSA, Sigma‐Aldrich) for 1 hr. The membranes were incubated with individual primary antibody (Table S1 ) at 4°C overnight. Then the membranes were incubated with corresponding HRP‐conjugated secondary antibody (Table S1 ) at 25°C for 1 hr. The bands were detected with ECL chemiluminescence (Thermo Scientific, Waltham, MA, USA) and viewed in Fusion FX Vilber Lourmat (France).
9
Whole Cell Lysate Extraction and Co-IP
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The whole cell lysate was extracted using the RIPA buffer (Thermo Fisher, Waltham, MA). For immunoblotting, proteins (20 μg) were detected with specific primary antibodies and an HRP-conjugated secondary antibody. Antibody conjugates were visualized by the ECL chemiluminescence (Thermo Fisher). The Co-IP assays were performed as described previously 19 (link). Antibodies were used for immunoprecipitation: HK2, VDAC1 or normal rabbit IgG. Immunocomplexes were resolved by SDS-PAGE, and co-immunoprecipitated proteins were detected via western blot.
10
Western Blot Analysis of Sea Urchin Immune Proteins
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Bacteria with bound SpTrf proteins or samples of the nickel column eluates were heated to 95°C in SDS lysis buffer and separated by electrophoresis on a 12% sodium dodecyl sulfate—polyacrylamide gel (SDS-PAGE), transferred to PVDF membranes, and evaluated with anti-SpTrf antibodies according to [46 (link), 49 (link)]. The sea urchin complement C3 homologue, SpC3, was detected using anti-SpC3-6H as described [53 (link), 64 (link), 65 (link)]. The antibody against SpC3 was raised to a 50 kDa recombinant fragment of the N terminal region of the SpC3 α chain [64 (link)]. Secondary antibodies were labeled with horse radish peroxidase, and blots were processed for ECL chemiluminescence (Thermo Fisher Scientific) followed by imaging with a ChemiDoc XRS+ (Bio-Rad Laboratories).
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