Enhanced chemiluminiscence

Manufactured by PerkinElmer

Enhanced chemiluminescence is a sensitive and versatile technique used for the detection and quantification of proteins and other analytes in various applications. It involves the use of a chemiluminescent substrate that emits light upon enzymatic reaction, allowing for the visualization and analysis of target molecules.

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Lab products found in correlation

Image lab software version 5, by Bio-Rad (3 mentions) Chemidoc mp imaging system, by Bio-Rad (3 mentions) Trans blot turbo transfer system, by Bio-Rad (3 mentions) Stain free sds page gel, by Bio-Rad (2 mentions) Tissuelyser, by Qiagen (1 mentions) Protease inhibitor, by Roche (1 mentions) Phosphatase inhibitor, by Roche (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Ripa buffer, by Merck Group (1 mentions) Calnexin, by Santa Cruz Biotechnology (1 mentions) P akt s473, by Santa Cruz Biotechnology (1 mentions) P egfr, by Santa Cruz Biotechnology (1 mentions) P her2, by Cell Signaling Technology (1 mentions) P erk1 2, by Santa Cruz Biotechnology (1 mentions) Cyclin d1, by Santa Cruz Biotechnology (1 mentions)

4 protocols using enhanced chemiluminiscence

Protein Expression Analysis in ALS

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Equal amounts of protein (10 µg) were resolved in 4–15% stain free SDS-PAGE gels (Bio-rad). OB and OT proteins derived from control and ALS subjects (Table 2) were electrophoretically transferred onto nitrocellulose membranes using a Trans-blot Turbo transfer system (up to 25 V, 7 min) (Bio-rad). Membranes were probed with primary antibodies (between 1:200 and 1:1000 dilution) in 5% nonfat milk or BSA according to manufacturer instructions. After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5000), the immunoreactivity was visualized by enhanced chemiluminiscence (Perkin Elmer) and detected by a Chemidoc MP Imaging System (Bio-Rad). Equal loading of the gels was assessed by stain free digitalization. After densitometric analyses (Image Lab Software Version 5.2; Bio-Rad), optical density values were normalized to total stain in each gel lane and expressed as arbitrary units.

Lachén-Montes M., Mendizuri N., Ausin K., Andrés-Benito P., Ferrer I., Fernández-Irigoyen J, & Santamaría E. (2020). Amyotrophic Lateral Sclerosis Is Accompanied by Protein Derangements in the Olfactory Bulb-Tract Axis. International Journal of Molecular Sciences, 21(21), 8311.

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Western Blot Protein Expression Analysis

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Equal amounts of protein (10 μg) were resolved in 12.5% SDS-PAGE gels. OB proteins derived from human samples were electrophoretically transferred onto nitrocellulose membranes using a Trans-blot Turbo transfer system (up to 25V, 7min) (Bio-rad). Equal loading of the gels was assessed by Ponceau staining. Membranes were probed with primary antibodies at 1:1000 dilution in 5% nonfat milk or BSA. After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5000), the immunoreactivity was visualized by enhanced chemiluminiscence (Perkin Elmer) and detected by a Chemidoc MP Imaging System (Bio-Rad). After densitometric analyses (Image Lab Software Version 5.2; Bio-Rad), optical density values were expressed as arbitrary units and normalized to GAPDH.

Lachén-Montes M., González-Morales A., Zelaya M.V., Pérez-Valderrama E., Ausín K., Ferrer I., Fernández-Irigoyen J, & Santamaría E. (2017). Olfactory bulb neuroproteomics reveals a chronological perturbation of survival routes and a disruption of prohibitin complex during Alzheimer’s disease progression. Scientific Reports, 7, 9115.

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Western Blotting of Protein Extracts

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Equal amounts of protein (10 µg) were resolved in 4-15% stain free SDS-PAGE gels (BioRad). Protein extracts derived from OBC1 cells were electrophoretically transferred onto nitrocellulose membranes using a Trans-blot Turbo transfer system (up to 25V, 7min) (BioRad). Membranes were probed with primary antibodies at 1:1000 dilution in 5% nonfat milk or BSA according to manufacturer instructions. After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5000), the immunoreactivity was visualized by enhanced chemiluminiscence (Perkin Elmer) and detected by a Chemidoc MP Imaging System (Bio-Rad). Equal loading of the gels was assessed using Stain-free imaging technology. Thus, protein normalization was performed by measuring total protein directly on the gels used for western blotting. After densitometric analyses (Image Lab Software Version 5.2; Bio-Rad), optical density values were expressed as arbitrary units and normalized to total protein levels.

Lachén-Montes M., Mendizuri N., Ausín K., Echaide M., Blanco E., Chocarro L., de Toro M., Escors D., Fernández-Irigoyen J., Kochan G, & Santamaría E. (2022). Metabolic dyshomeostasis induced by SARS-CoV-2 structural proteins reveals immunological insights into viral olfactory interactions. Frontiers in Immunology, 13, 866564.

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Comprehensive Protein Expression Analysis

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Flash-frozen tumor fragments were homogenized using a Tissuelyser (Qiagen) and lysed in RIPA buffer (Sigma) supplemented with 1X protease inhibitor (Roche) and phosphatase inhibitor (Roche) cocktails. Cells were washed with ice-cold PBS twice and lysed in RIPA buffer as described above. Lysates were gently rocked for 30 min at 4°C and centrifuged at 13,000 rpm for 15 min. Protein concentrations in supernatants were measured with the BCA protein assay (Pierce); 20 μg of total protein were fractionated by SDS-PAGE and transferred to nitrocellulose membranes (BioRad). Membranes were blocked with 5% non-fat dry milk and then incubated at 4°C overnight with the following primary antibodies: [from Cell Signalling Technologies: P-HER2 (#2249 1:1000), HER2 (#2242; 1:5000), P-HER3 (#4791; 1:1000), HER3 (#4754; 1:500), HER4 (#4795; 1:500), P-HER4 (#4757; 1:500), P-EGFR (#2237; 1:1000), EGFR (#2646; 1:5000),AKT (#9272; 1:10000), P-AKT_S473 (#9271; 1:500), P-ERK1/2 (#9101; 1:10000), ERK (#9102; 1: 10000), pRB (#9308; 1: 1000), and Calnexin (#2679; 1:10000)]; [from Santa Cruz Biotechnology: ERα (sc-8002; 1:1000) and cyclin D1 (sc-718; 1:200)]. nitrocellulose membranes were then incubated with HRP conjugated anti-rabbit or anti-mouse secondary antibodies for 1 h at room temperature and immunoreactive bands were detected by enhanced chemiluminiscence (Perkin Elmer).

Sudhan D.R., Schwarz L.J., Guerrero-Zotano A., Formisano L., Nixon M., Croessmann S., Ericsson P.I., Sanders M.E., Balko J.M., Avogadri-Connors F., Cutler RE J.r., Lalani A.S., Bryce R., Auerbach A, & Arteaga C.L. (2018). Extended adjuvant therapy with neratinib plus fulvestrant blocks ER/HER2 crosstalk and maintains complete responses of ER+/HER2+ breast cancers: Implications to the ExteNET trial. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(2), 771-783.

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