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Ecl prime detection

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ECL Prime detection is a chemiluminescent detection system for Western blotting. It is designed to provide sensitive and consistent detection of proteins on membranes.

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Lab products found in correlation

Anti ubiquitin, by Cell Signaling Technology (2 mentions) Anti rad6, by Abcam (2 mentions) Anti rps3 us3, by Cell Signaling Technology (2 mentions) Anti rpl11 ul5, by Cell Signaling Technology (2 mentions) Anti rps20 us10, by Thermo Fisher Scientific (2 mentions) Anti ubc13, by Novus Biologicals (2 mentions) Anti mouse igg2a, by Abcam (2 mentions) Pvdf membrane, by Thermo Fisher Scientific (2 mentions) Anti k63 ubiquitin, by Merck Group (2 mentions) Anti ha, by Thermo Fisher Scientific (2 mentions) Anti myc, by Thermo Fisher Scientific (2 mentions) Anti actin, by Cell Signaling Technology (2 mentions) Anti flag, by Merck Group (2 mentions) Anti puromycin, by Merck Group (2 mentions) Anti gapdh, by Abcam (2 mentions) Anti mouse igg, by Cytiva (2 mentions) Anti rabbit igg, by Cytiva (2 mentions) Protein a g plus agarose, by Santa Cruz Biotechnology (2 mentions) D pbs, by Wisent (1 mentions) Anti mouse, by Merck Group (1 mentions)

6 protocols using ecl prime detection

1

Western Blot of Ubiquitin Modifications

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Proteins were separated by standard 10 - 15% SDS-PAGE loaded in Laemmli buffer. Samples were transferred to PVDF membrane (ThermoFisher), and immunoblotting was performed using the following antibodies: anti-K63 ubiquitin (1:4,000; EMD Millipore, cat. no. 05-1308, clone apu3), anti-actin (1:6,000; Cell Signaling, cat. no. 4967), anti-GAPDH (1:4,000; Abcam, cat. no. ab9485), anti-Myc (1:5,000; ThermoFisher, cat. no. R950-25 and PA1-981), anti-Rad6 (1:8,000; Abcam, cat. no. ab31917), anti-Rps3/uS3 (1:6,000; Cell Signaling, cat. no. 9538S), anti-Rpl11/uL5 (1:6,000; Cell Signaling, cat. no. 18163S), anti-HA (1:10,000; ThermoFisher, cat. no. 71-5500), anti-Rps20/uS10 (1:9,000; ThermoFisher, cat. no. PA5-75383), anti-ubiquitin (1:10,000; Cell Signaling Technology cat. No. 3936S), anti-FLAG (1:3,000; MilliporeSigma, cat. no. F3165), anti-puromycin (1:4,000; MilliporeSigma, cat. no. MABE343), anti-ubc13 (1:1,000; Novus Biologicals, cat. no. NBP1-76593). Anti-mouse IgG (1:8,000-12,000; Cytiva, ca. no. NA931), anti-rabbit IgG (1:6,000-12,000; Cytiva, cat. no. NA934), and Anti-mouse IgG2a (1:10,000-1:12,000; Abcam, cat. no. ab97245) secondary antibodies conjugated with HRP and ECL Prime detection reagents were acquired from Cytiva. All antibodies have been validated by the manufacturer or are expected to react with the species used in this study based on sequence similarity.
Simões V., Cizubu B.K., Harley L., Zhou Y., Pajak J., Snyder N.A., Bouvette J., Borgnia M.J., Arya G., Bartesaghi A, & Silva G.M. (2022). Redox-sensitive E2 Rad6 controls cellular response to oxidative stress via K63-linked ubiquitination of ribosomes. Cell reports, 39(8), 110860.
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2

Western Blot of Ubiquitin Modifications

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Proteins were separated by standard 10 - 15% SDS-PAGE loaded in Laemmli buffer. Samples were transferred to PVDF membrane (ThermoFisher), and immunoblotting was performed using the following antibodies: anti-K63 ubiquitin (1:4,000; EMD Millipore, cat. no. 05-1308, clone apu3), anti-actin (1:6,000; Cell Signaling, cat. no. 4967), anti-GAPDH (1:4,000; Abcam, cat. no. ab9485), anti-Myc (1:5,000; ThermoFisher, cat. no. R950-25 and PA1-981), anti-Rad6 (1:8,000; Abcam, cat. no. ab31917), anti-Rps3/uS3 (1:6,000; Cell Signaling, cat. no. 9538S), anti-Rpl11/uL5 (1:6,000; Cell Signaling, cat. no. 18163S), anti-HA (1:10,000; ThermoFisher, cat. no. 71-5500), anti-Rps20/uS10 (1:9,000; ThermoFisher, cat. no. PA5-75383), anti-ubiquitin (1:10,000; Cell Signaling Technology cat. No. 3936S), anti-FLAG (1:3,000; MilliporeSigma, cat. no. F3165), anti-puromycin (1:4,000; MilliporeSigma, cat. no. MABE343), anti-ubc13 (1:1,000; Novus Biologicals, cat. no. NBP1-76593). Anti-mouse IgG (1:8,000-12,000; Cytiva, ca. no. NA931), anti-rabbit IgG (1:6,000-12,000; Cytiva, cat. no. NA934), and Anti-mouse IgG2a (1:10,000-1:12,000; Abcam, cat. no. ab97245) secondary antibodies conjugated with HRP and ECL Prime detection reagents were acquired from Cytiva. All antibodies have been validated by the manufacturer or are expected to react with the species used in this study based on sequence similarity.
Simões V., Cizubu B.K., Harley L., Zhou Y., Pajak J., Snyder N.A., Bouvette J., Borgnia M.J., Arya G., Bartesaghi A, & Silva G.M. (2022). Redox-sensitive E2 Rad6 controls cellular response to oxidative stress via K63-linked ubiquitination of ribosomes. Cell reports, 39(8), 110860.
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3

Evaluating Autophagy Markers in EBV-B Cells

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EBV-B cells were treated for 3 hours with PapMV nanoparticles or rapamycin, with or without pretreatment with 5 mM 3-MA. Cells were collected and washed Dulbecco’s phosphate-buffered saline (D-PBS, Wisent Bioproducts). Proteins were extracted in the presence of HALT proteinase/phosphatase inhibitors (ThermoFisher) from the above-mentioned pelleted cells and quantified by Bio-Rad protein assay. Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Membranes were incubated with rabbit an anti-human LC3B antibody (Ab; 1:3,000) (Novus Biologicals), a rabbit anti-ATG5-specific Ab (Cell Signaling) or a mouse anti-human β-actin-specific Ab (1:10,000) (Millipore). Membranes were revealed with horseradish peroxidase (HRP)-linked anti-rabbit (1:5,000) or anti-mouse (1:40,000 for β-actin) Abs (Chemicon) followed by enhanced chemiluminescence (ECL) prime detection (Amersham) on film. Densitometry was performed with ImageJ software and is reported as the ratio between densities of LC3-II over LC3-I bands or the ratio of ATG5 over β-actin bands.
Possamaï D., Hanafi L.A., Bellemare-Pelletier A., Hamelin K., Thébault P., Hébert M.J., Gagnon É., Leclerc D, & Lapointe R. (2021). MHC class I antigen cross-presentation mediated by PapMV nanoparticles in human antigen-presenting cells is dependent on autophagy. PLoS ONE, 16(12), e0261987.
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4

Western Blot Analysis of SK Channels

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The TH and PSD fractions were loaded equally (10 μg) and separated in 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. After blocking with 5% non-fat dry milk in TBS containing 0.1% Tween-20, membranes were incubated with SK1 (1:500, Alomone-labs), SK2 (1:500, Sigma-Aldrich) or SK3 (1:500, Alomone-labs) antibody overnight at 4 °C. The specific binding of these antibodies have been previously described [3 (link); 28 ; 33 (link)]. Then membranes were probed with anti-rabbit-HRP 1:5000 followed by Amersham ECL Prime detection. The intensity of each sample’s SK1, SK2 or SK3 band and its respective β-actin was measured using ImageJ software. For the analysis, intensity of each sample’s SK1, SK2 and SK3 band was normalized to its respective β-actin and represented as a percent of the average intensity of saline bands.
Hipólito L., Fakira A.K., Cabañero D., Blandón R., Carlton S.M., Morón J.A, & Melyan Z. (2015). In vivo activation of the SK channel in the spinal cord reduces the NMDA receptor antagonist dose needed to produce antinociception in an inflammatory pain model. Pain, 156(5), 849-858.
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5

Immunoprecipitation of HE4 and Annexin II

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Ice-cold RIPA buffer (1 ml) was added to ovarian cancer cells, followed by incubation at 4°C for 30 min. After centrifugation at 15,000 × g for 30 min at 4°C, supernatant fractions were collected and treated with anti-HE4 antibody (10 μl) (Santa Cruz, goat polyclonal) or anti-annexin II (Proteintech, mouse monoclonal) for 3 h at 4°C. Protein A/G PLUS-Agarose (20 μl; Santa Cruz)was added, followed by incubation on a rocker platform overnight at 4°C. The procedure was followed as described previously [20 (link)]. The negative control contained only 10 μl HE4 or ANXA2 antibody without protein. Immunoprecipitates were subsequently subjected to 12% SDS gel electrophoresis and analyzed via Western blot using HE4 monoclonal (Abcam, Rabbit) and annexin II monoclonal (Abcam, Mouse) antibodies. Proteins were visualized using ECL reagent (Amersham ECL Prime detection). Experiments were repeated three times.
Zhuang H., Tan M., Liu J., Hu Z., Liu D., Gao J., Zhu L, & Lin B. (2014). Human epididymis protein 4 in association with Annexin II promotes invasion and metastasis of ovarian cancer cells. Molecular Cancer, 13, 243.
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6

Immunoprecipitation of FOXA1 and HDAC3

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OC cells was treated with ice-cold RIPA buffer (1 ml) and incubation on ice for 30 min. The cells suspension was centrifuged at 12,000 g for 30 min at 4 °C, then discarded the supernatant and collected supernatant fractions. Anti-FOXA1 antibody (10 μl) (Abcam, rabbit monoclonal) or anti-HDAC3 antibody (CST, mouse monoclonal) was added in the supernatant fractions overnight at 4 °C. The mixture was treated with Protein A/G PLUS-Agarose (20 μl; Santa Cruz) and incubation them on the rocker platform overnight at 4 °C. The negative control was 10ul anti-Rabbit IgG (abcam). Western blot was used to analyze the immunoprecipitates with FOXA1 monoclonal (Abcam, Rabbit) and HDAC3 monoclonal (Abcam, Mouse) antibodies. ECL reagent (Amersham ECL Prime detection) was used to visualize the proteins.
Lou T., Liu C., Qu H., Zhang Z., Wang S, & Zhuang H. (2022). FOXA1 can be modulated by HDAC3 in the progression of epithelial ovarian carcinoma. Journal of Translational Medicine, 20, 19.
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