Protein extraction and Western blotting were performed as previously described [61 (link)]. Ezh2 (5246, Cell Signaling Technology, 1:100) and actin (sc-1615, Santa Cruz Biotechnology, Dallas, TX, USA, 1:5000) antibodies were used. Bands were visualized under the ECL Prime detection system (GE Healthcare, Chicago, Il, USA). Actin served as a loading control. The full length uncropped original Western blots were shown in the Supplementary information.
Ecl prime detection system
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Japan
The ECL Prime detection system is a laboratory equipment product designed for the detection of chemiluminescent signals. It functions by utilizing enhanced chemiluminescence technology to enable sensitive and quantitative analysis of target molecules in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Complete mini, by Roche (3 mentions)
Hybond p, by GE Healthcare (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Hybond lfp membrane, by GE Healthcare (2 mentions)
Anti β actin antibody, by GeneTex (2 mentions)
Cellytic m mammalian cell lysis extraction reagent, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Actin sc 1615, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti map2, by Abcam (1 mentions)
Anti s6 ribosomal protein, by Cell Signaling Technology (1 mentions)
Anti glua1, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc touch, by Bio-Rad (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Anti mouse igg secondary antibody, by Cell Signaling Technology (1 mentions)
Blue loading buffer pack, by Cell Signaling Technology (1 mentions)
21 protocols using ecl prime detection system
1
Protein Extraction and Western Blotting
2
Western Blotting of Key Neuronal Signaling Proteins
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Neurons were washed in PBS and lysed in RIPA buffer (150 mM NaCl, 50 mM HEPES, 0.5% NP40, 1% Sodium-deoxycholate). After 1 h under mild agitation, the lysate was clarified by centrifugation for 20 min at 16,000 g. All procedures were performed at 4°C. Protein samples were measured via standard Bradford assay (Bio-Rad., Hercules, CA, USA). For protein identification and relative quantification via Western blotting, a proper volume of sample containing an equal amount of proteins was diluted with 0.25% 5× Laemmli buffer and loaded onto 10% SDS-PAGE gels; the proteins were transferred onto nitrocellulose membrane (Sigma-Aldrich) at 80 V for 120 min at 4°C.
The primary antibodies were applied overnight in a blocking buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20 and 5% non-fat dry milk); primary antibodies (source in parentheses) included: anti-ERK1/2, anti-P-ERK1/2, anti-AKT, anti-P-AKT, anti- synapsin1–3, anti-GluA1, anti-β-actin, anti-s6 ribosomal protein (Cell Signaling); anti-MAP2 (Abcam). Proteins were detected using the ECL prime detection system (GE Healthcare). Images were acquired with the imaging system ChemiDoc Touch (BioRad) and protein quantification was performed measuring the optical density of the specific bands with ImageLab software (BioRad).
The primary antibodies were applied overnight in a blocking buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20 and 5% non-fat dry milk); primary antibodies (source in parentheses) included: anti-ERK1/2, anti-P-ERK1/2, anti-AKT, anti-P-AKT, anti- synapsin1–3, anti-GluA1, anti-β-actin, anti-s6 ribosomal protein (Cell Signaling); anti-MAP2 (Abcam). Proteins were detected using the ECL prime detection system (GE Healthcare). Images were acquired with the imaging system ChemiDoc Touch (BioRad) and protein quantification was performed measuring the optical density of the specific bands with ImageLab software (BioRad).
3
Hypoxia-Induced PDK1 Expression Analysis
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After 24 hours culture under normoxia or hypoxia, 1 × 106 cells of NB4 or THP-1 were lysed in 0.1 ml Blue Loading Buffer Pack (Cell Signaling #7722, Danvers, MA) and 0.02 ml of lysed cells were electrophoresed in 7.5% polyacrylamide gel. The gel was electroblotted onto hybond-P (GE Healthcare, UK). The membrane was hybridized with antibody to PDK1 (mouse monoclonal antibody, IgG1: abcam #ab110335, UK) at 1:1000 dilution and reacted with anti-mouse IgG secondary antibody (Cell Signaling #7076, Danvers, MA) at 1:2000 dilution. The membrane was visualized by ECL-prime Detection System (GE Healthcare, UK). β-actin (horseradish peroxidase-conjugated rabbit monoclonal antibody, IgG: Cell Signaling #5125, Danvers, MA) was also tested as control.
4
Western Blot Analysis of Protein Expression
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Twenty-four hours after transfection with siRNAs, the cells were lysed with RIPA buffer (Nakarai Tesque). Samples were boiled for 10 min in NuPAGE sample buffer (Invitrogen) and were separated by SDS–polyacrylamide gel electrophoresis. The proteins were subsequently transferred onto an Immobilon-P PVDF transfer membrane (Millipore, Bedford, MA). The membranes were blocked with 3% bovine albumin (Nakarai Tesque) prepared in phosphate-buffered saline with Tween 20 (PBS-T) and then incubated with one of the primary antibodies: β-actin (1:10,000, Sigma-Aldrich, St. Louis, MO), PAX3 (1:500, R&D Systems, Minneapolis, MN), Myogenin (1:500, Novus Biologicals, Centennial, CO), and B7-H3 (1:1000, Cell Signaling Technology, Danvers, MA). The membranes were then washed with PBS-T and incubated with anti-mouse or anti-rabbit secondary antibody (1:10,000, Santa Cruz Biotechnology, Santa Cruz, CA). Antibody binding was detected using an ECL prime detection system (GE Healthcare, Buckinghamshire, UK).
5
Western Blot Analysis of FoxM1 Expression
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After treatment with CQ (20 and 30 μM), NQ (10 and 20 μM), thiostrepton (10 μM), or DMSO, cells were harvested and lysed in RIPA buffer (Cell Signaling Technology, Beverly, MA, USA) in the presence of protease inhibitor cocktail (Cell Signaling Technology). The protein concentration was determined using Bio-Rad protein assay (Bio-Rad). Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% nonfat milk and incubated with FoxM1 antibody (Cell Signaling Technology) at 4°C overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated antirabbit antibody (Cell Signaling Technology) for 2 hours at room temperature. Signals were detected using the ECL Prime detection system (GE health-care, Bangkok, Thailand), and β-actin detected with specific antibody (Sigma-Aldrich) was used as the loading control.
6
Protein extraction and detection in meiotic cells
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Whole cell extracts were prepared from 5 ml cells (4 × 107 cells/ml) sampled from the meiotic time course. Cells were lysed in 150 µl of lysis buffer (1.85 M NaOH, 7.5% β-mercaptoethanol at natural pH) and incubated for 10 min on ice. 150 μl of cold 50% trichloroacetic acid was added and incubated for a further 10 min on ice. The precipitated proteins were pelleted, resuspended in 100 µl of 2× Laemmli buffer, and neutralized by adding 5–10 µl of unbuffered 1 M Tris. Separation and blotting were performed according to standard procedures. Spp1-myc and Spp1-GFP proteins were detected using the 9E10 mouse anti-myc (ab56; Abcam) and anti-GFP (ab290; Abcam) primary antibodies and HRP-conjugated goat anti-mouse (1:100,000; Amersham) secondary antibody. Detection was performed by an ECL prime detection system (GE Healthcare).
7
Isolation and Immunoblotting of Streptococcal Cell Wall
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Streptococcal cell wall extracts were isolated as previously described26 (link). Briefly, overnight S. pyogenes cultures were diluted 1:10 in fresh THB and grown to an A600 of 0.4–0.8. The cells were pelleted and resuspended in 1/50th volume of 10 mM Tris-HCl (pH 8.0) containing 30% raffinose, 100 U/ml mutanolysin, 1 mg/ml lysozyme and 10% Protease Inhibitor Cocktail Set III (Calbiochem), and incubated for 3 h at 37 °C with occasional inversion. The cells were pelleted and the supernatant (cell wall extract) was dialysed into PBS overnight and concentrated by centrifugation (10,000 MWCO). For immunoblotting, proteins were separated on 7% tris-acetate (TA) gels (Invitrogen) and transferred to Hybond LFP membranes (GE Healthcare). Membranes were blocked with 5% skimmed milk prior to the addition of 4 μg/ml IVIG (Endobulin®, Baxter). Bound antibodies were detected using a 1:80,000 dilution of HRP-conjugated goat anti human IgG (Sigma-Aldrich) and the ECL prime detection system (GE Healthcare).
8
TRPM4 Protein Expression Analysis in H9c2 Cells
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Cultured H9c2 cells were harvested, washed in cold PBS, and lysed with the Ambion KDalert Lysis Buffer (Life Technologies). Protein concentration was measured with the Protein Assay Rapid Kit (Wako, Osaka, Japan). Equal amounts of protein from each sample were separated by 8% SDS-PAGE (Bolt bis-tris Plus Gel, Life Technologies), and transferred onto a polyvinylidene difluoride (PVDF) membrane (Invitrogen, Tokyo, Japan). The non-specific sites were blocked by incubating the membrane (1 h, room temperature) in 3% (w/v) bovine serum albumin (BSA) added to Tris-buffered saline containing 0.005% (v/v) Tween-20 (TBS-T). Then, the membrane was incubated overnight (4°C) with TRPM4 antibody (1:250; ab63080, Abcam). The membrane was washed in TBS-T, and incubated with HRP-conjugated secondary antibody (1:50000) for 1 h. The membrane was washed with TBS-T, and specific proteins were visualized using an ECL Prime Detection System (GE Healthcare, Tokyo, Japan).
9
Chlamydomonas Protein Extraction and Immunoblotting
10
Quantitative Analysis of Surfactant Protein D
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