Ecl plus western blot detection kit

Manufactured by Cytiva

Sourced in United States, United Kingdom

The ECL Plus Western blot detection kit is a chemiluminescent detection system used for the visualization of protein bands on Western blots. It provides a sensitive and efficient method for the detection of low-abundance proteins.

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Lab products found in correlation

Protease inhibitor, by Roche (2 mentions) Radioimmunoprecipitation assay (ripa), by Merck Group (2 mentions) β actin, by Abcam (2 mentions) Ab76956, by Abcam (2 mentions) Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions) Bca protein kit, by Beyotime (1 mentions) Hrp conjugated goat anti rabbit, by Santa Cruz Biotechnology (1 mentions) Hrp conjugated goat anti mouse, by Santa Cruz Biotechnology (1 mentions) Polyvinylidene fluoride membrane, by Merck Group (1 mentions) Anti β actin, by Abcepta (1 mentions) Dnmt3a, by Cell Signaling Technology (1 mentions) Irak m, by Abcam (1 mentions) Total stat6, by Cell Signaling Technology (1 mentions) β tubulin, by Bioworld Technology (1 mentions) Anti casp 1, by R&D Systems (1 mentions) Anti il 1β, by Santa Cruz Biotechnology (1 mentions) Anti nlrp3, by Adipogen (1 mentions) Bca kit, by Beyotime (1 mentions) Ab214821, by Abcam (1 mentions) Ab68418, by Abcam (1 mentions)

Close spelling variants of this product

ECL kit (545 citations) ECL Plus (368 citations) ECL detection kit (245 citations) ECL Plus kit (186 citations)

10 protocols using ecl plus western blot detection kit

Immunoprecipitation and Western Blot Analysis of BTG2 and KLF15

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We immunoprecipitated the total proteins from the liver extract with antibodies against BTG2 and KLF15 (Santa Cruz Biotechnology) using protein A/G PLUS-Agarose (Santa Cruz Biotechnology). The immunoprecipitated proteins were then subjected to Western blot analysis using the corresponding antibodies^{11 (link)}. Signals were developed using an ECL-Plus Western blot detection kit (Amersham Bioscience, Piscataway, NJ, USA).

Kim Y.D., Hwang S.L., Jeon H.J., Jeon Y.H., Nedumaran B., Kim K, & Lee S.E. (2019). B-cell translocation gene 2 enhances fibroblast growth factor 21 production by inducing Kruppel-like factor 15. Scientific Reports, 9, 3730.

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Quantitative Western Blot Analysis Protocol

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Western blotting was carried out as described previously (Lin et al., 2019 (link); Wu et al., 2019 (link)). Briefly, the proteins from each cell layer extract were quantified using the BCA Protein kit (Beyotime Biotechnology, Shanghai, China). Protein extracts were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). Membranes were blocked with 5% non-fat milk in Tris–buffered saline for 1 h at room temperature. The membranes were incubated with primary antibodies, including anti-RAP2a (1:2,000; catalog no. NBP2-24574; Novusbio), DNMT3a (1:500; catalog no. 2160; Cell Signaling Technology), and anti-β-actin (1:3,000; catalog no. AP53385; Abgent) at 4°C overnight, followed by incubation with HRP-conjugated goat anti-rabbit (1:5,000; catalog no. sc-2004; Santa Cruz Biotechnology; RRID:AB_631746) or HRP-conjugated goat anti-mouse (1:5,000; sc-2005; Santa Cruz Biotechnology; RRID:AB_631746) secondary antibodies at 37°C for 1 h. The immunoreactive bands were visualized by using the ECL Plus Western Blot Detection kit (Amersham Biosciences United Kingdom), and densitometric quantification of band intensity from three independent experiments was carried out with the Image-Pro Plus 6.0 software. The relative expression level of the target protein was normalized to the intensity of the β-actin band.

Wu F., Lin X., Shan S.K., Li F., Xu F., Zhong J.Y., Guo B., Zheng M.H., Wang Y., Mo Z.H, & Yuan L.Q. (2020). The Suppression of miR-199a-3p by Promoter Methylation Contributes to Papillary Thyroid Carcinoma Aggressiveness by Targeting RAP2a and DNMT3a. Frontiers in Cell and Developmental Biology, 8, 594528.

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Western Blot Analysis of Lung Macrophages

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Lung macrophages were lysed in buffer containing RIPA (Sigma) supplemented with protease inhibitors (Roche Diagnostics) and gels were subjected to electrophoresis as previously described (26 ). Membranes were and incubated with antibodies against IRAK-M (Abcam; 1:1000), total-STAT6 (cell signaling,1:500), phosphor-STAT6 (Abcam, 1:500) or β-actin (Abcam, 1:10, 000). Signals were developed with an ECL Plus Western blot detection kit (Amersham, Arlington Heights, IL). Densitometry was calculated using Image J.

Ballinger M.N., Newstead M.W., Zeng X., Bhan U., Mo X.M., Kunkel S.L., Moore B.B., Flavell R., Christman J.W, & Standiford T.J. (2015). IRAK-M promotes alternative macrophage activation and fibroproliferation in bleomycin-induced lung injury. Journal of immunology (Baltimore, Md. : 1950), 194(4), 1894-1904.

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BHK Cell Preparation and VLP Analysis

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To prepare cell samples, BHK cells were trypsinized from transfected wells, pelleted, and 100,000 cells were resuspended in 100 μl of 1x SDS loading buffer. Cells were boiled for 5 minutes and clarified by centrifugation at 10,000 × G prior to loading onto 12% SDS-PAGE gels. For experiments examining released VLPs, transfected cells were incubated as above, and at 48 hrs the cells and supernatant were harvested. The 100K pellet was derived from media collected from the cell culture that was initially centrifuged at 10K at 4°C for 20 min to clarify large debris, followed by centrifugation of the clarified media at 100,00 × G at 4°C for 1 hr. Pelleted VLPs were resuspended in 20ul of 1x Laemmli SDS sample buffer and protein was resolved on SDS-PAGE gels. Resolved proteins were transferred to nitrocellulose membranes. Membranes were blocked with 2% non-fat dry milk in phosphate buffered saline supplemented with 0.1% Tween 20. Membranes were incubated with polyclonal anti-hemaglutanin antibody (Abcam), followed by incubation with horse-peroxidase-conjugated anti-rabbit secondary antibody. Proteins were detected with the ECL Plus Western blot detection kit (Amersham) on a Storm 860 phosphorimager.

Steel J.J, & Geiss B.J. (2015). A Novel System for Visualizing Alphavirus Assembly. Journal of virological methods, 222, 158-163.

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Western Blot Analysis of NLRP3, Caspase-1, and IL-1β

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We obtained lysates and separated proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred them to polyvinylidene difluoride (PVDF) membranes. Blots were incubated with primary antibodies (anti-NLRP3 [AdipoGen Life Sciences, San Diego, California, US], anti-CASP-1 [R&D Systems, Inc., Minneapolis, Minnesota, US], anti-IL-1β [Santa Cruz Biotechnology, Dallas, Texas, US] or β-tubulin [bioWORLD, Dublin, Ohio, US]) overnight at 4°C. Incubation with secondary anti-rabbit horseradish peroxidase (HRP) or anti-goat HRP (Hangzhou MultiSciences [Lianke] Biotech Co., Ltd., Hangzhou, China) was performed at room temperature (RT) for 1 h. We used an Electrochemiluminescence (ECL) Plus Western Blot Detection Kit (Amersham Biosciences [GE Healthcare, Chicago, Illinois, US]) for antibody detection.

Yang X., Zhan N., Jin Y., Ling H., Xiao C., Xie Z., Zhong H., Yu X., Tang R., Ma J., Guan J., Yin G., Wu G., Lu L, & Wang J. (2021). Tofacitinib restores the balance of γδTreg/γδT17 cells in rheumatoid arthritis by inhibiting the NLRP3 inflammasome. Theranostics, 11(3), 1446-1457.

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Western Blot Analysis of Extracellular Vesicle Proteins

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The expression of proteins was determined by western blot as previously described (Lin et al., 2018 (link)). Briefly, the concentration of protein was detected using a BCA kit (Beyotime, Shanghai, China). 30 μg total protein was loaded onto 8% or 12% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with primary antibody at 4°C for over 12 h after blocking with 5% non-fat milk for 1 h. Subsequently, horseradish peroxidase (HRP)-conjugated goat–anti-rabbit (sc-2004, 1:5,000, Santa Cruz) or HRP-conjugated goat–anti-mouse (sc-2005, 1:5,000, Santa Cruz) secondary antibodies were used to incubate with the membrane at RT for 1 h. The immunoreactive bands were visualized using the ECL Plus Western Blot Detection Kit (Amersham Biosciences U.K. Ltd.). The relative expression levels of proteins were normalized to the intensity of the β-actin band. Primary antibodies including CD9 (ab92726, 1:1,000, Abcam), CD63 (ab68418, 1:1,000, Abcam), CD81 (ab79559, 1:1,000, Abcam), Runx2 (ab76956; 1:1,000, Abcam), Fetuin-A (16571-1-AP, 1:1,000, Proteintech), MGP (10734-1-AP, 1:1,000, Proteintech), Annexin-A2 (11256-1-AP, 1:1,000, Proteintech), BMP-2 (ab214821, 1:1,000, Abcam), Rankl (23408-1-AP, 1:500, Proteintech), and β-actin (ab6276, 1:3,000, Abcam).

Lin X., Zhu T., Xu F., Zhong J.Y., Li F., Shan S.K., Wu F., Guo B., Zheng M.H., Wang Y., Xu Q.S., Liao X.B., Lu H.Y., Xie X.B, & Yuan L.Q. (2021). Plasma Exosomes Derived From Patients With End-Stage Renal Disease and Renal Transplant Recipients Have Different Effects on Vascular Calcification. Frontiers in Cell and Developmental Biology, 8, 618228.

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Protein Quantification in Lung Cells

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Lungs were harvested from infected and treated animals as described and homogenized to a single cell suspension. Cells were lysed in buffer containing RIPA (Sigma) supplemented with protease inhibitors (Roche Diagnostics). For immunoblot analysis, 20 μg protein was loaded onto 10% SDS-PAGE gels, subjected to electrophoresis, and transferred to membranes (Millipore). Membranes were incubated with Abs against PVL (1:1,000; Abcam) or β-actin (1:10,000; Abcam). Signals were developed with an ECL Plus Western blot detection kit (Amersham, Arlington Heights, IL).

Bhan U., Podsiad A.B., Kovach M.A., Ballinger M.N., Keshamouni V, & Standiford T.J. (2015). Linezolid Has Unique Immunomodulatory Effects in Post-Influenza Community Acquired MRSA Pneumonia. PLoS ONE, 10(1), e0114574.

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Protein Expression Analysis by Western Blot

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Protein expression analysis was detected by Western blot analysis as previously described [29 (link), 40 (link)]. The membranes were incubated with primary antibodies, including anti-BMF (ab9655, 1:1000, Abcam), anti-Runx2 (ab76956, 1:1000, Abcam), anti-p16 (10883-1-AP, 1:1000, Proteintech), anti-p21 (10355-1-AP, 1:1000, Proteintech), and anti-GAPDH (sc-365062, 1:3000, Santa Cruz Biotechnology) at 4°C overnight, followed by incubation with the horseradish peroxidase-conjugated goat anti-rabbit (sc-2004, 1:5000, Santa Cruz Biotechnology) or goat anti-mouse (sc-2005, 1:5000, Santa Cruz Biotechnology) secondary antibodies at 37°C for 1 hour. The immunoreactive bands were visualized using the ECL Plus Western blot detection kit (Amersham Biosciences U.K. Ltd) and densitometric quantification of band intensity from three independent experiments was carried out with the Image-Pro Plus 6.0 software. The relative expression level of target protein was normalized to the intensity of the GAPDH band.

Lin X., Zhan J.K., Zhong J.Y., Wang Y.J., Wang Y., Li S., He J.Y., Tan P., Chen Y.Y., Liu X.B., Cui X.J, & Liu Y.S. (2019). lncRNA-ES3/miR-34c-5p/BMF axis is involved in regulating high-glucose-induced calcification/senescence of VSMCs. Aging (Albany NY), 11(2), 523-535.

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Immunoprecipitation and Western Blot Analysis

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Immunoprecipitation and Western blot assays were performed according to the previously described procedures [4 (link)]. Antibodies Runx2 (Abcam, UK, 1:1000), ALP (Abcam, UK, 1:2000), SM22ɑ (Abcam, UK, 1:1000), p16 (Proteintech, USA, 1:500), p21 (Cell signaling, USA, 1:1000), LC3-II (Abcam, UK, 1:500), SQSTM1 (Abcam, UK, 1:1000), Atg10 (Abcam, UK, 1:1000), Bhlhe40 (Proteintech, USA, 1:500), GAPDH (Abcam, UK, 1:4000), and β-actin (Proteintech, USA, 1:2000) were used in this study. HA-VSMCs were transfected as indicated and lysed in an immunoprecipitation buffer. The lysates were centrifuged for 20 min. Supernatants were incubated with specific antibodies overnight at 4℃ and protein A/G Agarose beads for 4 h. The immunoprecipitates were washed three times with PBS, and then analyzed via SDS-PAGE. The protein bands were visualized using ECL-Plus Western blot detection kit (Amersham BioSciences, UK).

Li S., Ni Y., Li C., Xiang Q., Zhao Y., Xu H., Huang W., Wang Y., Wang Y., Zhan J, & Liu Y. (2022). Long noncoding RNA SNHG1 alleviates high glucose-induced vascular smooth muscle cells calcification/senescence by post-transcriptionally regulating Bhlhe40 and autophagy via Atg10. Journal of Physiology and Biochemistry, 79(1), 83-105.

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Bradykinin-induced eNOS Activation Assay

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BCAEC were purchased from Cell Applications, Inc. Dulbecco’s Modified Eagle’s Media (DMEM), fetal bovine serum (FBS), antibiotic and antimicotic solutions were from Gibco®. DMEM Nonessential Amino Acids and Hank’s Balanced Salt Solution (HBSS) phenol red free were from Mediatech, Cellgro Inc. Protease, and phosphatase inhibitor cocktails, dimethyl sulfoxide (DMSO) and bradykinin (BK) were obtained from Sigma Aldrich. β-tubulin, phospho-eNOS (p-eNOS Ser1177) and eNOS primary antibodies, as well as the anti-rabbit horseradish peroxidase (HRP) conjugated secondary antibody were from Cell Signaling Technology. Polyvidinil fluoride (PVDF) transfer membrane was from Millipore. Enhanced chemiluminescence (ECL) Plus Western Blot detection kit was from Amersham. The nitrite/nitrate fluorometric assay kit was from Cayman Chemical.

Varela C.E., Fromentin E., Roller M., Villarreal F, & Ramirez-Sanchez I. (2015). Effects of a natural extract of Aronia Melanocarpa berry on endothelial cell nitric oxide production. Journal of food biochemistry, 40(4), 404-410.

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