Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed with 10%–15% sodium dodecyl sulfate-polyacrylamide gel and 50 µg of cell lysate was loaded in each well. After electrophoresis, the cells were electro-transferred to Immobilon-P transfer membrane (Millipore, Billerica, MA, USA) under 200 mA for 2 hours. Three percent of BSA (AMRESCO, Solon, OH, USA) was used to inhibit nonspecific binding. After that, the primary antibody was added, reacted at room temperature for 1 hour, washed and secondary antibody was used. One of the ECL peroxidase-labeled anti-rabbit (Amersham, Piscataway, NJ, USA) or ECL peroxidase-labeled anti-mouse (Amersham) was selected for the primary antibody host, and then sensitized using Amersham ECL Western Blotting Detection Reagents (Amersham).
Amersham ecl western blotting detection reagent
Manufactured by Cytiva
Sourced in United States, United Kingdom
The Amersham ECL western blotting detection reagent is a chemiluminescent substrate used in Western blotting techniques to detect and visualize proteins. It is designed to provide a sensitive and reliable method for protein detection.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p transfer membrane, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Polyvinylidene fluoride (pvdf), by Thermo Fisher Scientific (1 mentions)
Anti manf antibody, by Abcam (1 mentions)
Las 3000, by Cytiva (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Hrp linked goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Hrp conjugated donkey anti goat igg, by Santa Cruz Biotechnology (1 mentions)
Mini trans blot electrophoretic transfer cell apparatus, by Bio-Rad (1 mentions)
Alphaeasefc software, by Bio-Techne (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Complete mini edta free protease inhibitor, by Roche (1 mentions)
Immobilon pvdf membrane, by Merck Group (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Amersham imager 680, by GE Healthcare (1 mentions)
Imagequant tl software version 7, by GE Healthcare (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Anti flag monoclonal antibody, by Merck Group (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Mouse monoclonal anti β actin, by Santa Cruz Biotechnology (1 mentions)
14 protocols using amersham ecl western blotting detection reagent
1
Western Blot Quantification Protocol
2
Quantifying TREM2 and MANF Proteins in Stroke
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Western blot analysis was performed at 24 hours after MCAo from the ipsilateral cortex and subcortical regions. Brain protein abundance was determined by Bio‐Rad Assay Method. A total of 20‐25 μg of equivalent protein was loaded on NuPAGE 4%‐12% Bis‐Tris gels and run at 125 V for 120 minutes, and then, proteins were transferred onto PVDF (Invitrogen) paper. TREM2 and MANF proteins were detected using anti‐TREM2 (Millipore‐Sigma) and anti‐MANF antibody (Abcam). Finally, TREM2 and MANF proteins were imaged in Fujifilm LAS‐3000 using Amersham™ ECL™ Western Blotting Detection Reagents. TREM 2 and MNAF protein bands were quantified by Multigage software, and the results were expressed as TREM2/GAPDH and MANF/GAPDH ratio, respectively.
3
Western Blot Analysis of Immune Signaling Proteins
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Cell lysates were collected in 1×RIPA buffer (Cell Signaling Technology, Inc., Danvers, MA) supplemented with Complete Mini, EDTA-free protease inhibitor (Roche Applied Science, Indianapolis, IN). Protein concentration was assayed by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Proteins were resolved on 4–20% SDS-PAGE gel and transferred to PVDF membrane using Mini Trans-Blot Electrophoretic Transfer Cell apparatus (Bio-Rad, Hercules, CA). Primary antibodies used for western blot analysis were rabbit monoclonal RIG-I (D14G6), cleaved CASP3 (Asp175) (5A1E), cleaved PARP (Asp214) (D64E10) XP (Cell Signaling technology, Inc., Danvers, MA), IRF3 antibody [EP2419Y] (Abcam, Cambridge, MA), and goat polyclonal anti-actin (I-19) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Secondary antibodies used were HRP-linked goat anti-rabbit IgG (Cell Signaling technology, Inc., Danvers, MA) and HRP-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immune complexes were visualized with Amersham ECL Western Blotting Detection Reagents and exposed to X-ray films (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) according to the manufacturer’s instruction. The protein level was quantitated by densitometry measurement using AlphaEaseFC software (Alpha Innotech, Miami, FL).
4
Immunoblotting of Borrelia Proteins
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Four weeks after tick feedings, blood was collected from mice and immunoblotting was performed as previously described [17] (link). Protein lysates from 1×107 spirochetes and 1 µg of recombinant Borrelia immunogenic protein A (rBipA) were analyzed using Mini-PROTEAN TGX precast gels (Bio-RAD, Hercules, CA). Gels were transferred to Immobilon PVDF membrane (Millipore, Billerica, MA, USA). Immunoblots were probed for one hour with mouse sera diluted at 1∶500 and the secondary molecule HRP-rec-Protein A (Life Technologies) was applied at a 1∶4,000 dilution for one hour. Serological reactivity was determined by chemiluminescence using the Amersham ECL Western Blotting Detection Reagents (GE Healthcare Bio-Sciences Corp., Piscataway, NJ).
5
Western Blot Analysis of Cell Lysates
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Cell lysates were prepared as described elsewhere in this report. Whole cell lysates were prepared as previously described44 (link). The amount of proteins in cell lysates was measured by CBB solution for Protein Assays (Nacalai Tesque). An equal amount of cell lysates was separated by SDS-PAGE. Gels were transferred to 0.22-µm ClearTrans® nitrocellulose membranes (FUJIFILM Wako Pure Chemical Corporation). After blocking with 5% skim milk in 0.5% Tween 20–PBS (PBS-T) overnight, membranes were treated with primary antibodies in 5% skim milk–PBS-T for 1 h and washed with PBS-T. The membranes were further incubated with secondary antibodies in 5% skim milk–PBS-T for 1 h and then washed with PBS-T. Protein bands were visualized using Amersham™ ECL Western Blotting Detection Reagents (Cytiva, Tokyo, Japan) or ImmunoStar® Zeta (FUJIFILM Wako Pure Chemical Corporation), acquired by Amersham™ Imager 680 (GE Healthcare Japan, Tokyo, Japan) and analyzed by ImageQuant™ TL software version 7.0 (GE Healthcare Japan).
6
FLAG-tagged Arabidopsis Protein Extraction
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Arabidopsis proteins were extracted with a buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.1% Triton X‐100, 1 mM dithiothreitol, 2 mM NaF and protease inhibitor cocktail (Roche). The protein samples were electrophoresed through a 10% SDS‐PAGE gel, and the protein was electrotransferred onto an Immobilon P membrane (Millipore Sigma). Immunodetection was performed using a 1:5000 dilution of an anti‐FLAG monoclonal antibody (Millipore Sigma). The blot was then hybridized with horseradish peroxidase‐conjugated secondary antibodies and visualized with Amersham ECL Western Blotting Detection Reagents (Cytiva).
7
Western Blot Analysis of Serglycin
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Cells were lysed with RIPA-buffer containing Protease Inhibitor Cocktail (Sigma), incubated for 30 min on ice, centrifuged for 15 min at 13000g. The protein concentration was quantified using Bradford Bio-Rad Protein Assay (BioRad). Total proteins (15 μg) were treated with NuPAGE LDS Sample Buffer (Life Technologies) containing 10% β-mercaptoethanol for 5 min at 96°C, resolved in 10% SDS-PAGE gels and transferred to nitrocellulose membranes Amersham Protran 0.45 NC (Amersham). The membranes were blocked with 5% milk in PBST (0,1%) for 1 h and incubated with primary antibodies [mouse anti-serglycin monoclonal (Santa Cruz, 1:200) or mouse anti-β-Actin monoclonal (Santa Cruz), 1:500] overnight at 4°C followed by secondary goat anti-mouse IgG (H+L)-HRP peroxidase-conjugated antibodies (BioRad) for 2 h at RT. Proteins were detected with an Amersham ECL Western Blotting Detection Reagent (Amersham) according to the manufacturer's instructions.
8
Western Blot Analysis of Protein Lysates
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293T cells were lysed with cell lysis buffer (25 mM Tris–Cl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF, 5% glycerol, 0.5% Triton X-100, and protease inhibitors [Roche, Indianapolis, IN, USA]). The small intestines were rinsed with PBS and homogenized in lysis buffer (1% NP40, 150 mM Tris–Cl, 50 mM NaCl, 1 mM EDTA). The cell or tissue lysates were separated with 8–15% gradient SDS-PAGE and transferred into PVDF membrane (Amersham-Pharmacia Biotech, Piscataway, NJ, USA). The proteins were probed with an anti-Flag monoclonal antibody diluted 1:4000 and an anti-β-actin antibody diluted 1:10,000. The immune complexes were detected with peroxidase-conjugated goat anti-mouse IgG antibody (Santa Cruz Biotechnology) and visualized with Amersham ECL western blotting detection reagent (Amersham-Pharmacia Biotech).
9
LL Inhibits RANKL-Induced Osteoclastogenesis
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RAW 264.7 cells were seeded in a 60mm cell culture dish at the density of 5 × 105 cells per well in α-MEM medium. After 24 hours, the cells were incubated with α-MEM supplemented with RANKL (100 ng/mL) and LL of various concentration (1, 10, and 100 μg/mL) for 24 hours. The cells were lysed in RIPA buffer. Equal amounts of proteins were separated by SDS-PAGE and transferred using an electro transfer kit to nitrocellulose membranes (Whatman, UK). The membranes were blocked with 5% skim milk; probed for 24 hours with the specific primary antibodies such as c-fos and NFATc1, at 4°C; and then incubated with horseradish peroxidase conjugated secondary antibodies for 1 hour. Immunoreactivities were detected using an Amersham™ ECL™ Western blotting detection reagent (Buckinghamshire, UK).
10
Western Blot Analysis of Tumor Cell Proteins
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For western blotting, tumour cells were washed and lysed with CellLytic M (Sigma-Aldrich) containing protease and phosphatase inhibitors. The lysate was cleared by centrifugation at 15,000 rpm for 15 min and denatured at 95 °C for 5 min. The lysate was stored at −80 °C until immunoblot analysis. Proteins were separated by electrophoresis using a NuPAGE 4–12% Bis-Tris Gel (Thermo Fisher Scientific) and transferred to Amersham Protran (Cytiva). After blocking the membrane with 5% skim milk for 1 h, it was incubated overnight at 4 °C with specific primary antibodies. After hybridisation with secondary antibodies, the protein bands were visualised using Amersham ECL western blotting detection reagent (Cytiva). The antibodies used in this study are listed as follows; anti-EPCAM (#ab223582, Abcam), anti-SNAP25 (#5308, Cell Signaling Technology), anti-Actin (#MA5-11869, Invitrogen), anti-rabbit IgG (#7074, Cell Signaling Technology), and anti-mouse IgG (#7076, Cell Signaling Technology).
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