Pierce ecl detection kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Pierce ECL detection kit is a chemiluminescent detection system designed for the sensitive visualization of proteins on Western blots. The kit contains the necessary reagents for enhanced chemiluminescence (ECL) detection, allowing for the detection of low-abundance proteins.

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Lab products found in correlation

Hybond ecl nitrocellulose membrane, by GE Healthcare (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Hybond, by Cytiva (2 mentions) Pvdf membrane, by GE Healthcare (1 mentions) Phosphatase inhibitor, by Roche (1 mentions) P jnk, by Cell Signaling Technology (1 mentions) Bcl xl, by Cell Signaling Technology (1 mentions) Bcl 2, by Cell Signaling Technology (1 mentions) Goat anti mouse igg antibody, by Abcam (1 mentions) Goat anti mouse secondary antibody, by Abcam (1 mentions) Anti β actin antibody, by Abcam (1 mentions) Goat anti rabbit secondary antibody, by Abcam (1 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions) Western blotting gels, by Bio-Rad (1 mentions) Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Alexa 488 or 555 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions) Centrin 2, by Santa Cruz Biotechnology (1 mentions) Aurora a, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

ECL kit (865 citations) ECL detection kit (265 citations) Pierce ECL (169 citations) Pierce ECL kit (48 citations)

8 protocols using pierce ecl detection kit

SDS-PAGE and Western Blotting

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Protein samples were diluted in SDS (sodium dodecyl sulfate) loading buffer. Protein concentration was determined by amido black assays. Samples were separated by SDS-PAGE (polyacrylamide gel electrophoresis) and subsequently blotted on PVDF membranes (GE Healthcare). Immunoreactivity of primary antibodies (see Table 1) was detected using HRP-conjugated secondary antibodies and the Pierce^® ECL detection kit (Thermo Scientific).

Schoen M., Reichel J.M., Demestre M., Putz S., Deshpande D., Proepper C., Liebau S., Schmeisser M.J., Ludolph A.C., Michaelis J, & Boeckers T.M. (2016). Super-Resolution Microscopy Reveals Presynaptic Localization of the ALS/FTD Related Protein FUS in Hippocampal Neurons. Frontiers in Cellular Neuroscience, 9, 496.

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Western Blot Analysis of Apoptosis Markers

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Cells were lysed in RIPA buffer containing protease inhibitor cocktail (Sigma) and phosphatase inhibitors (Roche Applied Bioscience, Indianapolis, IN, USA). Cell lysates were subjected to SDS-PAGE followed by immunoblot with corresponding antibodies. Polyclonal antibodies against JNK1, JNK2, pJNK, Bcl-XL, Bcl-2, BAX, phospho threonine and COX IV were from Cell Signaling Technology (Beverly, MA, USA; all used at 1:1000). Actin monoclonal antibody (1:5000) was from Sigma. ALDH1L1 and Bid were detected using in-house polyclonal antibodies (1:20 000) generated against full-length proteins. Rabbit polyclonal phospho-Bid-specific (pT59) antibody was generated by AnaSpec (Freemont, CA, USA) against ELQ(pT)DGNRSSHSRLG peptide (1:3000). In all experiments, a Hybond TM-ECL nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburg, PA, USA) and Pierce ECL detection kit (Thermo Scientific, Waltham, MA, USA) were used.

Prakasam A., Ghose S., Oleinik N.V., Bethard J.R., Peterson Y.K., Krupenko N.I, & Krupenko S.A. (2014). JNK1/2 regulate Bid by direct phosphorylation at Thr59 in response to ALDH1L1. Cell Death & Disease, 5(7), e1358-.

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Western Blot Analysis of ALDH1L1 in NIH3T3 Cells

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NIH3T3 cells were lysed in RIPA buffer containing 1x protease inhibitor cocktail (Sigma). Cell lysates were subjected to SDS-PAGE (20 μg of the total protein loaded per lane) followed by Western blot assays with corresponding antibodies. ALDH1L1 was detected using in-house rabbit polyclonal antibody (1:10000) generated against purified recombinant rat ALDH1L1 [7 (link), 16 (link)]. Primary antibodies used in this study are listed in S1 Table. All in-house antibodies have been verified in our previous studies [5 (link), 17 (link), 18 (link)]. HRP-conjugated secondary goat anti-rabbit IgG antibody and goat anti-mouse IgG antibody were from Abcam (both used at 1:7000). A Hybond TM-ECL nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburg, PA, USA) and Pierce ECL detection kit (ThermoFisher Scientific, Waltham, MA, USA) were used.

Khan Q.A., Pediaditakis P., Malakhau Y., Esmaeilniakooshkghazi A., Ashkavand Z., Sereda V., Krupenko N.I, & Krupenko S.A. (2018). CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G0/G1 to S-phase. PLoS ONE, 13(7), e0199699.

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Western Blot Analysis of KIFC1

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36 h after transfection, cells were scraped from culture plates and sonicated in lysate buffer with 1X protease inhibitor cocktail (Invitrogen). Polyacrylamide gel electrophoresis was performed for protein resolution, and then transfer was made to a polyvinylidene difluoride membrane. The membrane was incubated in 1:1,000 mouse monoclonal antibody specific for KIFC1 (M-63: sc-100947) from Santa Cruz Biotechnology (Dallas, TX, USA) with 1:10,000 goat anti-mouse secondary antibody (Abcam; Cambridge, MA, USA). Β-actin was used as an internal loading control and was detected using rabbit monoclonal anti-Β-actin antibody with goat anti-rabbit secondary antibody (both from Abcam). The Pierce ECL detection kit (Thermo Scientific; Waltham, MA, USA) was used to visualize bands.

Ogden A., Garlapati C., Li X.(., Turaga R.C., Oprea-Ilies G., Wright N., Bhattarai S., Mittal K., Wetherilt C.S., Krishnamurti U., Reid M.D., Jones M., Gupta M., Osan R., Pattni S., Riaz A., Klimov S., Rao A., Cantuaria G., Rida P.C, & Aneja R. (2017). Multi-institutional study of nuclear KIFC1 as a biomarker of poor prognosis in African American women with triple-negative breast cancer. Scientific Reports, 7, 42289.

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Western Blotting Analysis of Cytochrome P450 Enzymes

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All Western blotting gels, membranes and electrophoresis equipment were purchased from Bio-Rad Laboratories. Total protein (20–30 µg) from treated HPHs were separated on SDS/PAGE (10% gels) and electrophoretically transferred on to PVDF membranes. Subsequently, membranes were blocked for 1 h at room temperature (24 °C) with 5 % non-fat dried skimmed milk powder in 0.1% TBS-T (Tris-buffered saline containing 0.1% Tween 20) before the incubation of primary antibody diluted in blocking buffer. Mouse monoclonal antibodies against human CYP2B6 and CYP3A4 were provided by Dr Frank Gonzalez (Laboratory of Metabolism, National Cancer Institute/National Institute of Health, Bethesda, MD, U.S.A.) and were diluted to 1:1000 for overnight incubation at 4°C. Anti-β-actin antibody (1:1000 dilution; Santa Cruz Biotechnology) was included as an internal control. The membranes were then washed three times with 0.1% TBS-T buffer and incubated with the appropriate secondary HRP (horseradish peroxidase)-conjugated antibody diluted to 1:4000–5000 in 2% non-fat dried skimmed milk powder in 0.1 % TBS-T for 2 h at room temperature. The membranes were developed using the Pierce ECL detection kit (Thermo Scientific).

Chen T., Laurenzana E.M., Coslo D.M., Chen F, & Omiecinski C.J. (2014). Proteasomal interaction as a critical activity modulator of the human constitutive androstane receptor. The Biochemical journal, 458(1), 95-107.

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Immunoblotting and Immunofluorescence of Cell Proteins

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Protein lysates were prepared from ~70% confluence cells and frozen tissue samples (sonicated). Briefly, PAGE was used to resolve the proteins and transferred on to PVDF membrane (Millipore). Pierce ECL detection kit (Thermo Scientific) was used to visualize the immune-reactive bands corresponding to respective primary antibodies. β-actin was used as loading control. For immunofluorescence staining, cells were grown on glass coverslips and fixed with ice cold methanol for 10 min. Blocking was done by incubating with 2% bovine serum albumin/PBS.0.05% Triton X-100 at 37°C for 1 h. Primary Antibodies against γ-tubulin, α-tubulin were incubated with coverslips for 1 h at 37°C at the dilution 1:2000. The cells were washed with 2% bovine serum albumin/PBS for 10 min at room temperature before incubating with a 1:2000 dilution of Alexa 488- or 555-conjugated secondary antibodies Invitrogen (Carlsbad, CA, USA). Antibodies against γ- tubulin, α-tubulin and vimentin were from Sigma (St. Louis, MO, USA), Aurora-A, Plk4 and β-actin were from cell signaling, Centrin-2 and Cyclin E from Santacruz and PeriCentrin-2 from Abcam. Horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Pannu V., Mittal K., Cantuaria G., Reid M.D., Li X., Donthamsetty S., McBride M., Klimov S., Osan R., Gupta M.V., Rida P.C, & Aneja R. (2015). Rampant centrosome amplification underlies more aggressive disease course of triple negative breast cancers. Oncotarget, 6(12), 10487-10497.

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Lipid Binding Assay for ApoL6

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Membrane lipid strips ((p-6002) Echelon biosciences) were blocked in buffer containing PBS, 0.1% Tween 20, 3% BSA for 1 h. The strips were then probed with ApoL6-GST for 4 h. The strips were washed 3 times with a buffer containing PBS and 0.1% Tween20 and then probed with GST antibody followed by anti-rabbit HRP conjugate as the secondary antibody. The strips were developed with Pierce ECL detection kit (Thermo-Fisher).

Wang Y., Nguyen H.P., Xue P., Xie Y., Yi D., Lin F., Dinh J., Viscarra J.A., Ibe N.U., Duncan R.E, & Sul H.S. (2024). ApoL6 associates with lipid droplets and disrupts Perilipin1-HSL interaction to inhibit lipolysis. Nature Communications, 15, 186.

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Membrane Protein Separation and Detection

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Inner and outer membranes were isolated by membrane fractionation as described above and resuspended in Laemmli SDS sample buffer (5% β-mercaptoethanol, 3% [wt/vol] SDS, 10% glycerol, 0.02% bromophenol blue, 63 mM Tris-Cl [pH 6.8]) (69 (link)), and boiled for 5 min. Samples were electrophoresed in quadruplicate (10%) SDS-polyacrylamide gels for separation. Proteins from three gels were transferred to a 0.45-μm-pore-size nitrocellulose membrane (GE Healthcare) and incubated with either rabbit polyclonal anti-IcsA antibody (Edwin Oaks, Walter Reed Army Institute of Research) diluted 1:10,000, rabbit polyclonal anti-SecA antibody (Donald Oliver, Wesleyan University) diluted 1:10,000, or rabbit polyclonal anti-OmpA antibody (Donald Oliver, Wesleyan University) diluted 1:5,000. Proteins were detected using horseradish peroxidase-conjugated goat anti-rabbit antibody (diluted 1:5,000). Signal was detected by developing the blot with Pierce ECL detection kit (Thermo Fisher). Proteins from the fourth gel were visualized by Coomassie brilliant blue staining and used to assess equal loading of samples for immunoblotting.

Rossi R.M., Yum L., Agaisse H, & Payne S.M. (2017). Cardiolipin Synthesis and Outer Membrane Localization Are Required for Shigella flexneri Virulence. mBio, 8(4), e01199-17.

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