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Western bright ecl

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Western Bright ECL is a chemiluminescent substrate used for the detection of proteins in Western blotting applications. It provides a sensitive and quantitative way to visualize proteins that have been separated by gel electrophoresis and transferred to a membrane.

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Lab products found in correlation

Mini protean tetra system, by Bio-Rad (3 mentions) Chemidoc xrs gel imaging system, by Bio-Rad (3 mentions) Streptavidin peroxidase conjugate, by Merck Group (3 mentions) Roti block, by Carl Roth (3 mentions) Amersham ecl prime blocking reagent, by GE Healthcare (1 mentions) Nupage transfer buffer, by Thermo Fisher Scientific (1 mentions) Nativepage novex 3 12 bis tris protein gels, by Thermo Fisher Scientific (1 mentions) Nativepage sample buffer, by Thermo Fisher Scientific (1 mentions) Amersham ecl rabbit igg, by GE Healthcare (1 mentions) Pvdf membrane, by Merck Group (1 mentions)

6 protocols using western bright ecl

1

Biotinylated Tyrosine Labeling Assay

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Example 9

See FIG. 27

Labeling reactions were performed in a 150 μL solution consisting of 20 mM MES/K pH 7.0, 100 mM KCl, 10 mM MgCl2, 2.5 mM ATP, 1 mM tyrosine-biotin 12, 1 μM TTL, 5 μM nanobody and 5 mM DTT. The mixture was incubated at 37° C. for 20 h. Proteins were separated by SDS-PAGE and wet blotted onto a nitrocellulose membrane using a Bio-Rad Mini-Protean Tetra System (250 mA, 1 h). The membrane was blocked with Roti-Block (Carl Roth, Karlsruhe, Germany) for 1 h at ambient temperature and incubated for 1 h with streptavidin peroxidase conjugate (Merck Millipore, Darmstadt, Germany) (1:2000) at ambient temperature. Immunodetection was performed with WesternBright chemiluminescence solution (Western Bright ECL, Biozym Scientific, Hessisch Oldendorf, Germany) and a ChemiDoc™ XRS+ gel imaging system (Bio-Rad, Hercules, Calif., US). See FIG. 22.

US11401298B2. Means and methods for site-specific functionalization of polypeptides (2022-08-02). LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN [DE], FORSCHUNGSVERBUND BERLIN E.V. [DE]. Inventors: Heinrich Leonhardt [DE], Jonas Helma [DE], Dominik Schumacher [DE], Christian Hackenberger [DE].
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2

Tyrosine Biotinylation and Immunodetection

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Example 9

See FIG. 27

Labeling reactions were performed in a 150 μL solution consisting of 20 mM MES/K pH 7.0, 100 mM KCl, 10 mM MgCl2, 2.5 mM ATP, 1 mM tyrosine-biotin 12, 1 μM TTL, 5 μM nanobody and 5 mM DTT. The mixture was incubated at 37° C. for 20 h. Proteins were separated by SDS-PAGE and wet blotted onto a nitrocellulose membrane using a Bio-Rad Mini-Protean Tetra System (250 mA, 1 h). The membrane was blocked with Roti-Block (Carl Roth, Karlsruhe, Germany) for 1 h at ambient temperature and incubated for 1 h with streptavidin peroxidase conjugate (Merck Millipore, Darmstadt, Germany) (1:2000) at ambient temperature. Immunodetection was performed with WesternBright chemiluminescence solution (Western Bright ECL, Biozym Scientific, Hessisch Oldendorf, Germany) and a ChemiDoc™ XRS+ gel imaging system (Bio-Rad, Hercules, Calif., US). See FIG. 22.

US10745437B2. Means and methods for site-specific functionalization of polypeptides (2020-08-18). LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN [DE], FORSCHUNGSVERBUND BERLIN E.V. [DE]. Inventors: Heinrich Leonhardt [DE], Jonas Helma [DE], Dominik Schumacher [DE], Christian Hackenberger [DE].
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3

Assessing AQP4 Mutant OAP Assembly

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All AQP4 mutant constructs were studied for OAP assembly by using blue native gel electrophoresis (BN-PAGE). Therefore, HEK-293A cells were transfected as described before. Cells were lysed in 1X NativePageTM Sample Buffer (Invitrogen, # BN2003) supplemented with 500 mM 6-aminohexanoic acid, 1 % Triton X-100 and protease inhibitor cocktail, incubated for 30 min on ice and centrifuged at 22,000×g for 30 min. Supernatants were supplemented with NativePageTM 5 % G-250 (Invitrogen, #BN2004) and loaded to NativePAGE™ Novex™ 3-12 % Bis-Tris Protein Gels (Invitrogen, # BN1001). The running buffers were prepared according to the manufacturer’s protocol. Proteins were blotted onto polyvinylidene difluoride (PVDF) membrane using NuPage Transfer Buffer (Invitrogen, #NP0006). For immunoblot analysis, proteins were fixed for 15 min in 8 % acetic acid and membranes were blocked in 1 % Amersham ECL Prime Blocking Reagent (GE Healthcare, # RPN418) diluted in 0.1 % PBS-Tween. Primary rabbit anti-AQP4 antibody (Sigma, # A5971) was incubated at 4 °C overnight. Secondary antibody Amersham ECL Rabbit IgG (GE Healthcare, # Na934) was incubated at RT for 1 h. Labeled proteins were detected using the WesternBright ECL (Biozym, # 541004) and visualized on the Fusion FX7 Vilber Lourmat imaging system.
Tuller F., Holzer H., Schanda K., Aboulenein-Djamshidian F., Höftberger R., Khalil M., Seifert-Held T., Leutmezer F., Berger T, & Reindl M. (2016). Characterization of the binding pattern of human aquaporin-4 autoantibodies in patients with neuromyelitis optica spectrum disorders. Journal of Neuroinflammation, 13, 176.
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4

Cell Lysis and Protein Detection

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Total cell extracts were prepared using alkaline lysis followed precipitation with trichloroacetic acid (TCA) (Knop et al., 1999 (link)). Proteins were dissolved in SDS sample buffer (50 mM Tris-Cl pH 6.8, 10 mM EDTA, 5% glycerol, 2% SDS, 0.01% bromphenol blue) and analyzed by SDS-PAGE. Semi-dry western blotting was used to transfer proteins onto PVDF membranes (Merck Millipore). Immunodetection was performed using appropriate antibodies. Immunoreactive species were visualized using WesternBright ECL (Biozym) and detected using the LAS-4000 Fuji Imager.
Dederer V., Khmelinskii A., Huhn A.G., Okreglak V., Knop M, & Lemberg M.K. (2019). Cooperation of mitochondrial and ER factors in quality control of tail-anchored proteins. eLife, 8, e45506.
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5

Cell Lysis and Immunoblotting Protocol

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Cells were washed with 1× PBS and lysed in ice-cold Nonidet P40 lysis buffer (40 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.5% (v/v) Nonidet-P40, 10 mM β-glycerophosphate, 10 mM NaF, 1 mM EDTA, 1 mM PMSF, 1 μg/mL pepstatin, 1 μg/mL aprotinin, and 1 μg/mL leupeptin) for 10 min on ice. Upon centrifugation at 10,000× g and 4 °C, cleared lysates were normalized, resolved by SDS-PAGE, transferred onto a nitrocellulose membrane (Amersham Protran Premium 0.2 µm NC, Global Life Sciences Solutions Operations UK Ltd., Little Chalfont, UK), and immunoprobed with the antibodies indicated according to manufacturer’s recommendations. Enhanced chemiluminescence detection (Western Bright ECL, Advansta, Biozym Scientific GmbH, Oldendorf, Germany) was used in immunoblot assays.
Leitner P.D., Jakschitz T., Gstir R., Stuppner S., Perkams S., Kruus M., Trockenbacher A., Griesbeck C., Bonn G.K., Huber L.A, & Valovka T. (2022). Anti-Inflammatory Extract from Soil Algae Chromochloris zofingiensis Targeting TNFR/NF-κB Signaling at Different Levels. Cells, 11(9), 1407.
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6

Tyrosine-Biotin Labeling Reaction Protocol

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Example 9

See FIG. 27

Labeling reactions were performed in a 150 μL solution consisting of 20 mM MES/K pH 7.0, 100 mM KCl, 10 mM MgCl2, 2.5 mM ATP, 1 mM tyrosine-biotin 12, 1 μM TTL, 5 μM nanobody and 5 mM DTT. The mixture was incubated at 37° C. for 20h. Proteins were separated by SDS-PAGE and wet blotted onto a nitrocellulose membrane using a Bio-Rad Mini-Protean Tetra System (250 mA, 1h). The membrane was blocked with Roti-Block (Carl Roth, Karlsruhe, Germany) for 1 h at ambient temperature and incubated for 1 h with streptavidin peroxidase conjugate (Merck Millipore, Darmstadt, Germany) (1:2000) at ambient temperature. Immunodetection was performed with WesternBright chemiluminescence solution (Western Bright ECL, Biozym Scientific, Hessisch Oldendorf, Germany) and a ChemiDoc™ XRS+ gel imaging system (Bio-Rad, Hercules, Calif., US). See FIG. 22.

US10208084B2. Means and methods for site-specific functionalization of polypeptides (2019-02-19). LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN [DE], FORSCHUNGSVERBUND BERLIN E.V. [DE]. Inventors: Heinrich Leonhardt [DE], Jonas Helma [DE], Dominik Schumacher [DE], Christian Hackenberger [DE].
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