Luminol

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

Luminol is a chemical compound commonly used in forensic science and analytical chemistry. It is a chemiluminescent substance that emits light when it reacts with certain oxidizing agents, such as hydrogen peroxide and certain metal ions. Luminol is primarily used for the detection of trace amounts of blood at crime scenes, as it can reveal the presence of even small quantities of blood that may not be visible to the naked eye.

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Lab products found in correlation

Protein inhibitor cocktail, by Merck Group (3 mentions) Phorbol 12 myristate 13 acetate pma, by Merck Group (3 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) Hcn2 and hcn3, by Abcam (2 mentions) Secondary immunoglobulin g igg conjugated with horseradish peroxidase, by Santa Cruz Biotechnology (2 mentions) Cellytic mt mammalian tissue lysis extraction reagent, by Merck Group (2 mentions) Enhancer for western blotting, by Thermo Fisher Scientific (2 mentions) Goat anti rabbit, by Thermo Fisher Scientific (2 mentions) Horseradish peroxidase, by Merck Group (2 mentions) Reader infinite m200pro, by Tecan (2 mentions) Nunctm f96 microwelltm polystyrol plate, by Thermo Fisher Scientific (2 mentions) I control 1, by Tecan (2 mentions) Biotin maleimide, by Merck Group (1 mentions) Dbco peg4 biotin, by Merck Group (1 mentions) N succinimidyl s acetylthioacetate sata, by Merck Group (1 mentions) Dabcyl lpetg edans, by AnaSpec (1 mentions) Talon superflow, by GE Healthcare (1 mentions) Spectramax l, by Molecular Devices (1 mentions) Phe pro arg chloromethylketone ppack, by Prolytix (1 mentions) Spectramax m2, by Molecular Devices (1 mentions)

26 protocols using luminol

Luminol-based Oxidative Burst Assay

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An independent ethics committee approved the blood sample collection for neutrophil oxidative burst assay and MAT (Ethical Committee for Research at Masaryk University, Brno, Czechia, approval no. EKV-2018-083). All donors gave their written consent.
The oxidative burst (ROS production) of blood phagocytes was determined in diluted whole blood by luminol-enhanced chemiluminescence (CL) using an LM-01T microplate luminometer (Immunotech, Czech Republic). Briefly, the reaction mixture consisted of 6 μL of whole blood in 54 µl of RPMI-1640 growth medium mixed with 60 µl of dressing extract in physiological saline or an FBS-supplemented medium. This mixture was incubated at 37 °C for 20 min. Just before the start of the measurement, we added 1 mM luminol (a stock solution of 10 mM luminol in a 0.2 M borate buffer) (Molecular Probes, USA). We determined spontaneous ROS production, and ROS production induced by opsonised zymosan particles (OZP—0.1 mg/mL) (Sigma-Aldrich, USA) or phorbol 12-myristate 13-acetate (PMA—0.8 µM; Sigma-Aldrich, USA). Untreated samples without the tested extracts were evaluated as controls. The assays were run in duplicates. Chemiluminescence was recorded continuously for 90 min at 37 °C and was expressed as relative light units (RLU). We determined the total amount of ROS production from the integrated area under the chemiluminescence curve.

Nešporová K., Pavlík V., Šafránková B., Vágnerová H., Odráška P., Žídek O., Císařová N., Skoroplyas S., Kubala L, & Velebný V. (2020). Effects of wound dressings containing silver on skin and immune cells. Scientific Reports, 10, 15216.

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Western Blot Analysis of HCN Proteins

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Western blot analysis was carried out for different HCN proteins (1-4) in the bladder tissue. Briefly, bladder tissue was homogenized using CelLytic™ MT Mammalian Tissue Lysis/Extraction Reagent (Sigma, USA) in the presence of phenylmethylsulfonyl fluoride (1 mM), sodium orthovanadate (2 mM) and protein inhibitor cocktail (Sigma, USA). Protein estimation was done by BCA Protein Assay Kit (Pierce, Rockford, Illinois). An equal amount (50 μg/well) of denatured proteins was loaded in 10% tricine-SDS gel and blotted on polyvinylidene fluoride (PVDF) membranes using wet transfer system. After blocking (2 h at 37°C), membranes were incubated overnight at 4°C with primary antibodies specific for HCN1, HCN4 and Actin-β (Santa Cruz Biotechnology), HCN2 and HCN3 (Abcam, USA), in blocking buffer (pH 7.5). The membranes were then re-incubated for 2 h at room temperature with secondary immunoglobulin G (IgG)-conjugated with horseradish peroxidase (Santa Cruz Biotechnologies, USA). The blots were developed using luminol (Thermo Scientific, USA) and measured on Versa doc imaging system (Model 4000; BioRed, USA). Densitometry for protein specific band was done using AlphaEase FC StandAlone V. 4.0.0 software. β-Actin was used as an internal control to normalize the band density.

Kashyap M., Yoshimura N., Smith P.P., Chancellor M, & Tyagi P. (2015). Characterization of the role of HCN channels in β3-adrenoceptor mediated rat bladder relaxation. Bladder, 2(2), e15.

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Sortase A-Mediated Protein Labeling

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Biotin-maleimide, DBCO-PEG4-biotin and N-succinimidyl-S-acetyl-thioacetate (SATA) were from Sigma-Aldrich (Saint Louis, Missouri, USA). Luminol, Nunc 96-well maxisorp microtiter plates and Zeba Spin 7 kDa desalting columns were from Thermo Scientific (Waltham, Massachusetts, USA). Spectramax L and Spectramax M2 were from Molecular Devices (San Jose, California, USA). Streptavidin-polyHRP and secondary rabbit anti-mouse HRP antibody were from Agilent Technologies (Santa Clara, California, USA). BL21 pLyss E. Coli was obtained from Invitrogen (Carlsbad, California, USA) and vector pet30b-7M SrtA (plasmid # 51141), encoding a modified, calcium-independent Sortase A from S. aureus was a gift from Hidde Ploegh and ordered from Addgene (Watertown, Massachusetts, USA). DBCO-PEG (20 kDa) was from Jena Bioscience (Jena, Germany) and mPEG-maleimide (20 kDa) was from Broadpharm (Waples, San Diego, USA). DABCYL-LPETG-EDANS was from AnaSpec (Waddinxveen, the Netherlands). Gly₃-azide was from IRIS Biotech GmbH (Marktredwitz, Germany). Imidazole was from Merck Millipore (Burlington, Massachusetts, USA). Phe-Pro-Arg-chloromethylketone (PPACK) was from Haematologic Technologies (Essex Junction, Vermont, USA). Talon Superflow was from GE Healthcare (Hoevelaken, The Netherlands).

van Moorsel M.V., Urbanus R.T., Verhoef S., Koekman C.A., Vink M., Vermonden T., Maas C., Pasterkamp G, & Schiffelers R.M. (2019). A head-to-head comparison of conjugation methods for VHHs: Random maleimide-thiol coupling versus controlled click chemistry. International Journal of Pharmaceutics: X, 1, 100020.

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Quantifying Peroxidase Activity in Cells

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Peroxidase activity in neutrophils and adipose tissue was measured by the chemiluminescence assay using luminol plus near-infrared quantum dots, as previously described (18 (link)). Briefly, epididymal WAT and neutrophils were homogenized with RIPA buffer containing 1 mmol/L Na₃VO₄, 1 µg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride. Protein concentration was determined using the bicinchoninic acid method. The protein concentrations of homogenates were adjusted to 2 mg/mL and 1 mg/mL, respectively. Eighty microliters protein lysate were placed in a Costar 96-well black plate with 80 µL 2.3 mmol/L luminol (Thermo Fisher, Rockford, IL) and 1 µL 8 μmol/L QD800 (Invitrogen, Grand Island, NY). Then, 80 μL 2 mmol/L H₂O₂ were added to the mixtures to trigger production of HOCl. Luminescence was recorded for 20 seconds after the H₂O₂ addition to estimate peroxidase activity by using an M1000 microplate reader (Tecan Group Ltd., Männedorf, Switzerland).

Wang Q., Xie Z., Zhang W., Zhou J., Wu Y., Zhang M., Zhu H, & Zou M.H. (2014). Myeloperoxidase Deletion Prevents High-Fat Diet–Induced Obesity and Insulin Resistance. Diabetes, 63(12), 4172-4185.

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Western Blot Analysis of HCN Proteins

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Kashyap M., Yoshimura N., Smith P.P., Chancellor M, & Tyagi P. (2015). Characterization of the role of HCN channels in β3-adrenoceptor mediated rat bladder relaxation. Bladder, 2(2), e15.

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Immunoblotting Analysis of SMAR1 and β-Actin

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SMAR1 and β-Actin protein levels were determined by immunoblotting. In brief, the proteins were extracted using protein extraction buffer (20 mM Tris-HCl pH-7.8, 1 mM EDTA, 1 mM PMSF, 0.1% (v/v) Triton X-100, PI cocktail) and then quantified using Bradford’s reagent (Bio-Rad). Equal amount of protein was loaded on 10% SDS-PAGE and samples were electrotransferred to PVDF membrane at 100 V constant voltage. The blots were then saturated with 5% (w/v) BSA/3% (w/v) non-fat dry milk and reacted with respective primary antibodies, then incubated with secondary antibodies tagged with horseradish peroxidase. Signals were detected by chemiluminescence using luminol as a substrate (Thermo Scientific).

Mathai J., Mittal S.P., Alam A., Ranade P., Mogare D., Patel S., Saxena S., Ghorai S., Kulkarni A.P, & Chattopadhyay S. (2016). SMAR1 binds to T(C/G) repeat and inhibits tumor progression by regulating miR-371-373 cluster. Scientific Reports, 6, 33779.

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Antibody-based Analysis of Stress Response

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Antibiotics (penicillin, streptomycin), DMEM (Gibco, high glucose, L-glutamine), fetal calf serum (Gibco), and trypsin (10×, Gibco) were from Invitrogen (Life Technology, Waltham, MA, USA). Unless otherwise stated, chemicals were purchased from Sigma Aldrich (St. Louis, MO) or Mallinckrodt Baker B.V. (Deventer, NL). The proteasome inhibitor MG 132 was prepared as a 10 mmol/L stock-solution in DMSO is used. Primary antibodies: p53 (1C12) and phospho-p53 (Ser15) Abs (53 kDa; Cell Signaling, Essex, MA, USA, Mouse IgG, 1:500), p38 and phospho-p38 MAP kinase Abs (Thr180/Tyr182) (38 kDa; Cell Signaling, Rabbit IgG, 1:200), MDM2 Ab (90 kDa, R&D Systems, affinity-purified Rabbit IgG, 1:500), phosphorylated MDM2 Ab (Ser166) (pMDM2, Cell Signaling, Rabbit, IgG 1:500), and histone H3 Ab (FL-136) (Santa Cruz Biotechnology; Finnell Street, Dallas, TX, USA, Rabbit IgG, 1:250) were all prepared in NaAzid free blocking buffer. Stabilized peroxidase conjugated secondary antibodies: Goat Anti-Mouse (Thermo Fisher Scientific, Wyman Street, Waltham, MA, USA, 1:600) and Goat Anti-Rabbit (Thermo Scientific; 1:600) were prepared in NaAzid free blocking buffer. Luminol and enhancer for western blotting were from Thermo Scientific.

Lambert I.H., Enghoff M.S., Brandi M.L, & Hoffmann E.K. (2015). Regulation of p53 in NIH3T3 mouse fibroblasts following hyperosmotic stress. Physiological Reports, 3(6), e12412.

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Western Blot Analysis of Bladder Tissue

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Briefly, bladder tissue was homogenized using CellyticTM MT Mammalian Tissue Lysis Reagent (Sigma, USA) in presence of phenylmethylsulphonyl fluoride (PMSF, 1 mM), dithiothreitol (DTT, 2 mM), and protein inhibitor cocktail (Sigma, USA). After estimating protein content by BCA Protein Assay Kit (Pierce, Rockford, Illinois), 100 μg of denatured proteins were loaded in duplicate onto 10% SDS-polyacrylamide gel and blotted on PVDF membranes using a wet transfer system. After blocking with 5% skimmed milk (2h at RT), membranes were incubated overnight at 4°C with primary antibodies in blocking buffer followed by washing and re-incubation with HRP tagged secondary antibodies for 2h (Santa Cruz Biotechnologies, USA). The blots were developed using luminol (Thermo Scientific, USA) and measured on Versa doc imaging system (Model 4000; Bio Rad, USA) using Alpha Ease FC Stand Alone V. 4.0.0 software for normalizing the HCN band density of β-Actin as an internal control.

Singh N., Zabbarova I., Ikeda Y., Kanai A., Chermansky C., Yoshimura N, & Tyagi P. (2021). Role of Hyperpolarization-activated cyclic nucleotide-gated Channels in Aging Bladder Phenotype. Life sciences, 289, 120203.

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Stabilized Peroxidase-Conjugated Antibody Detection

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Stabilized peroxidase‐conjugated goat anti‐mouse (Thermo Scientific 1:5000) and Goat Anti‐Rabbit (Thermo Scientific; 1:5000). All antibodies were prepared in NaAzid free blocking buffer. Luminol and enhancer for western blotting were from Thermo Scientific.

Bach M.D., Sørensen B.H, & Lambert I.H. (2018). Stress‐induced modulation of volume‐regulated anions channels in human alveolar carcinoma cells. Physiological Reports, 6(19), e13869.

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Western Blot Analysis of Enzymatic Markers

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Cell lysis was done in RIPA buffer solution in the presence of protease inhibitors. The protein samples were separated by electrophoresis on 10% acrylamide gel and blotted to PVDF membrane. The immunoblotting assay blocking step was performed with 5% non-fat dry milk powder in 0.1% PBS Tween and incubated overnight with primary antibody for arginase 1 (Santa Cruz), iNOS (Santa Cruz) antibodies or alpha-tubulin (Sigma). The secondary antibody used was HRP-peroxidase (Life Technologies). The reaction was developed using Luminol (Thermo Scientific) and bands were quantified by Photoshop.

Cabanel M., Brand C., Oliveira-Nunes M.C., Cabral-Piccin M.P., Lopes M.F., Brito J.M., de Oliveira F.L., El-Cheikh M.C, & Carneiro K. (2015). Epigenetic Control of Macrophage Shape Transition towards an Atypical Elongated Phenotype by Histone Deacetylase Activity. PLoS ONE, 10(7), e0132984.

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