Chemiluminescent kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Chemiluminescent kit is a laboratory tool designed to detect and quantify specific biomolecules, such as proteins, through a luminescent-based assay. The kit provides the necessary reagents and components to perform this analysis.

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14 protocols using chemiluminescent kit

Protein Extraction and Western Blot

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RIPA lysis and extraction buffer were used to lyse cells on ice for 30 min as previously described (Niu et al., 2017 ). Samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to membranes. Membranes were blocked with 5% nonfat milk and incubated with primary antibodies at 4 °C overnight. Protein bands were detected by Chemiluminescent Kit (Thermo Scientific, USA).

Wang L., Qiu J.G., He J., Liu W.J., Ge X., Zhou F.M., Huang Y.X., Jiang B.H, & Liu L.Z. (2019). Suppression of miR-143 contributes to overexpression of IL-6, HIF-1α and NF-κB p65 in Cr(VI)-induced human exposure and tumor growth. Toxicology and applied pharmacology, 378, 114603.

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Western Blot Analysis of Fmo3 in Mouse Liver Microsomes

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Microsomes were isolated from livers as described previously (Cashman &Hanzlik, 1981 (link); Rudraiah et al., 2014 ) and stored at −80°C until use. Protein concentration was determined by the method of Lowry using Bio-Rad protein assay reagents (Bio-Rad Laboratories, Hercules, CA). For western blot analysis, microsomal proteins (10 μg) were electrophoretically resolved using 10% polyacrylamide gels and transferred onto PVDF-Plus membrane (Micron Separations, Westboro, MA). Membranes were blocked with 5 % non-fat powdered milk in tris buffered saline containing 0.05% tween-20(TBS-T) for 8 h. A rabbit anti-mouse Fmo3 primary antibody (GenScript USA Inc., NJ) (1:5000) was used to detect Fmo3 with β-actin as a loading control. Blots were then incubated with HRP conjugated secondary antibodies against rabbit IgG (1:2000) (Sigma-Aldrich, St Louis, MO). Protein-antibody complexes were detected using a chemiluminescent kit (Thermo Scientific, IL) with visualization using GeneMate blue autoradiography film (Bioexpress, Kaysville, UT). Precision plus protein dual color standards molecular weight marker (Bio-radLaboratories, Hercules, CA) was used to identify Fmo3 protein on the blot and microsomal protein isolated from naïve female mouse liver was used as a positive control.

Rudraiah S., Moscovitz J.E., Donepudi A.C., Campion S.N., Slitt A.L., Aleksunes L.M, & Manautou J.E. (2014). Differential Fmo3 Gene Expression in Various Liver Injury Models Involving Hepatic Oxidative Stress in Mice. Toxicology, 325, 85-95.

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Western Blot Analysis of Ischemia-Reperfusion Injury

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After ischemia-reperfusion injury, the cultured cells, or sliced brain tissue from infracted hemisphere, were dissected and homogenized in lysis buffer. The protein extracts were separated on SDS-polyacrylamide gel and electroblotted onto polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4℃ in a buffer with 0.1% Tween with primary antibodies specific to GLP-1 (Abcam, Cambridge, MA, USA), GLP-1R (Abcam), IB-1 (BD Transduction Laboratories, San Diego, CA, USA), phospho-SAPK/JNK (Cell Signaling Technology, Beverly, MA, USA), SAPK/JNK (Cell Signaling Technology), phospho-c-Jun (Cell Signaling Technology), p-MKK3/MKK6 (Cell Signaling Technology), phospho-p38 MAPK (Cell Signaling Technology), p38 (Cell Signaling Technology), p44/42 MAPK (Cell Signaling Technology), MMP-9 (Millipore), COX-2 (Cayman, Ann Arbor, MI, USA), β-actin (Sigma-Aldrich), and Transcription factor IID (TFIID) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Following this, membranes were then incubated with appropriate secondary antibodies. The signal was then detected using a chemiluminescent kit (Thermo Scientific, Rockford, IL, USA). Signals were normalized with β-actin or TFIID to standardize equal protein loading.

Kim S., Jeong J., Jung H.S., Kim B., Kim Y.E., Lim D.S., Kim S.D, & Song Y.S. (2017). Anti-inflammatory Effect of Glucagon Like Peptide-1 Receptor Agonist, Exendin-4, through Modulation of IB1/JIP1 Expression and JNK Signaling in Stroke. Experimental Neurobiology, 26(4), 227-239.

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Analyzing Protein Expression and Modifications

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Briefly, 30 μg of protein was lysed in ice-cold RIPA buffer with a protease inhibitor cocktail and phosphatase inhibitors (Sigma, MO, USA), loaded on a 12% Tris–glycine SDS polyacrylamide gel, and then transferred to a nitrocellulose membrane. Membranes were blocked with 5% milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 30 min, and then incubated at 4 °C overnight (1:1000) with primary antibodies: Catalase (PA5-29,183) and LC3BI, II (PA1-16,931) from Thermo Fisher, cleaved CASPASE 9 (20,750), CASPASE 3 (94,530), p62/SQSTM1 (5114), PARP (46D11), GAPDH (D16H11), and β-actin (8H10D10) from Cell Signaling, Ubiquitin (PA5-11,324), and LAMP1 (PA5-95,849) from Thermo Fisher. They were then incubated with goat anti-rabbit/mouse HRP (1:5000, Jackson Immunoresearch, West Grove, PA) for 1 h at room temperature and analyzed using a chemiluminescent kit (Thermo Scientific, Carlsbad, CA, USA). For immunoprecipitation, 50 μg of MVs were resuspended in RIPA buffer and incubated with Protein A/G agarose beads (Santa Cruz sc-2003) conjugated with CD63 antibodies (ab134045, Abcam) for 14 h at 4 °C. After washing and elution, all proteins were loaded onto a 15% SDS polyacrylamide gel.

Rupert C., Dell’ Aversana C., Mosca L., Montanaro V., Arcaniolo D., De Sio M., Bilancio A., Altucci L., Palinski W., Pili R, & de Nigris F. (2023). Therapeutic targeting of P2X4 receptor and mitochondrial metabolism in clear cell renal carcinoma models. Journal of Experimental & Clinical Cancer Research : CR, 42, 134.

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Immunoblotting Protocol with Antibody Detection

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Immunoblotting was performed as before (50 ). Membranes were incubated with primary antibodies as listed in Table S2. A secondary antibody conjugated to horseradish peroxidase (Pierce) and a chemiluminescent kit was used for detection (ThermoFisher).

Oster M., Galhuber M., Krstic J., Steinhoff J.S., Lenihan-Geels G., Wulff S., Kiefer M.F., Petricek K.M., Wowro S.J., Flores R.E., Yang N., Li C., Meng Y., Reinisch I., Sommerfeld M., Weger S., Habisch H., Madl T., Schulz T.J., Prokesch A, & Schupp M. (2022). Hepatic p53 is regulated by transcription factor FOXO1 and acutely controls glycogen homeostasis. The Journal of Biological Chemistry, 298(9), 102287.

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Western Blot Analysis of Histone Modifications

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Cells were harvested and lysed in ice-cold radio immunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitor cocktail and phosphatase inhibitors (Sigma, MO, USA). Protein concentrations were determined by BCA kit (Bio-Rad, Hercules, CA, USA). Briefly, 10 μg of protein were loaded on 12% tris-glycine polyacrylamide gel prior to being transferred to a PVDF membrane on ice. After transfer, membranes were blocked with 5% milk in tris-buffered saline with 0.1% Tween-20 (TBST) for 30 min. Membranes were then incubated with primary antibodies: anti-H3K36me3 (1:1000, Active Motif, Carlsbad, CA), anti-CS (1:1000, Cell Signaling, Danvers, MS), and anti-PGC1α (1:1000, Cell Signaling) at 4 °C overnight. After washing with TBST, membranes were incubated with goat anti-rabbit/mouse HRP (1:10,000, Jackson Immunoresearch, West Grove, PA) for 1 h at room temperature. After washing with TBST, membranes were observed by chemiluminescent kit (Thermo Scientific, Carlsbad, CA, USA).

Liu J., Hanavan P.D., Kras K., Ruiz Y.W., Castle E.P., Lake D.F., Chen X., O’Brien D., Luo H., Robertson K.D., Gu H, & Ho T.H. (2018). Loss of SETD2 induces a metabolic switch in renal cell carcinoma cell lines toward enhanced oxidative phosphorylation. Journal of proteome research, 18(1), 331-340.

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SDS-PAGE Western Blotting for pSTAT3 Quantification

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SDS-page Western blotting was used to examine the expression of pSTAT3 protein. Total protein content in the treated cells was extracted using Minute™ Total Protein Extraction Kit (Invent Biotechnologies, USA) as per manufacturer’s protocol. The concentration of protein samples was determined by a colorimetric assay using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA). A 25 μg of protein extract was separated by gradient (4–20%) sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane (Bio-Rad, USA) using a wet transfer unit (company name), blocked with 2.5% BSA in TBS-Tween buffer (0.12 M Tris-base, 1.5 M NaCl, 0.1% Tween 20) for an hour at room temperature, and incubated overnight at 4°C with the appropriate primary antibodies; β-actin (1:3000) and pSTAT3 (1:3000). This was followed by incubation with a HRP-conjugated goat anti-mouse secondary antibody (1:1000; Thermo Fisher Scientific, USA) for an hour and subsequent washing in TBST at RT. The blots were developed using Chemiluminescent kit (Thermo Fisher Scientific) to detect the target protein as per manufacturer’s protocol.

Misra S.K., Wu Z., Ostadhossein F., Ye M., Boateng K., Schulten K., Tajkhorshid E, & Pan D. (2019). Pro-nifuroxazide Self-Assembly Leads to Triggerable Nanomedicine for Anti-Cancer Therapy. ACS applied materials & interfaces, 11(20), 18074-18089.

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SDS-PAGE Immunoblotting Reagents

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Reagents for SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were from Bio-Rad Laboratories (Hercules, CA). Tris, aprotinin, adenosine triphosphate (ATP), dithiothreitol, phenylmethylsulfonyl fluoride, Triton X-100, Tween 20, glycerol, and bovine serum albumin (BSA) (fraction V) were from Sigma Chemical Co. (St. Louis, MO). Nitrocellulose paper (BA85, 0.2 mm) was from Schleicher & Schuell (Keene, NH). Ketamine hydrochloride was from Cristália (Itapira SP, Brazil). The chemiluminescent kit was from Thermo Fisher Scientific (Rockford, IL, USA). The following antibodies were used: Anti-IL6 (M-19) and anti–α-tubulin (B-7) were from Santa Cruz Biotechnology; anti-pERK1/2 (Thr²⁰²/Tyr²⁰⁴), anti-p44/42 MAPK (ERK1/2), anti-pACC (Ser⁷⁹), anti-ACC, anti–pAMPK (Thr¹⁷²), anti-AMPKα, and anti–α-tubulin were from Cell Signaling Technology. Secondary antibodies were from Thermo Fisher Scientific. Recombinant IL6 was from Calbiochem (San Diego, CA, USA). PD98059 was from LC Laboratory (Woburn, MA). Unless otherwise specified, routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The doses administered in each experimental group are given below.

Katashima C.K., de Oliveira Micheletti T., Braga R.R., Gaspar R.S., Goeminne L.J., Moura-Assis A., Crisol B.M., Brícola R.S., Silva V.R., de Oliveira Ramos C., da Rocha A.L., Tavares M.R., Simabuco F.M., Matheus V.A., Buscaratti L., Marques-Souza H., Pazos P., Gonzalez-Touceda D., Tovar S., del Carmen García M., Neto J.C., Curi R., Hirabara S.M., Brum P.C., Prada P.O., de Moura L.P., Pauli J.R., da Silva A.S., Cintra D.E., Velloso L.A, & Ropelle E.R. (2022). Evidence for a neuromuscular circuit involving hypothalamic interleukin-6 in the control of skeletal muscle metabolism. Science Advances, 8(30), eabm7355.

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Pseudorabies Virus Protein Expression

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PK15 or PI cells were infected at an MOI of 10 with PRV 151 or PRV 128 or mock infected and then harvested 16 h postinfection. Cell samples were lysed with radioimmunoprecipitation assay (RIPA)-light buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% SDS, 0.1% Triton X-100), protease inhibitor (Roche), and 100 mM dithiothreitol (DTT). Protein samples were heated to 70°C for 10 min, run on 4 to 12% SDS-PAGE gels, and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk–Tris-buffered saline supplemented with 0.1% Tween (TBS-T) for 1 h at room temperature and then incubated with a 1:200 dilution of rabbit anti-PRV EP0 (37 (link)), 1:1,000 mouse anti-PRV VP5 (38 (link)), 1:200 rabbit anti-PRV IE180 (12 (link)), and 1:10,000 mouse anti-β-actin (Sigma-Aldrich). Primary antibodies were washed with TBS-T, and membranes were incubated with 1:15,000 anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (KPL) for 1 h at room temperature. Signal was detected on radiographic film using a chemiluminescent kit (Thermo Scientific) according to the manufacturer’s instructions. The IE80 film was exposed longer than that of EP0 or VP5 to obtain comparable signal intensity.

Wu B.W., Engel E.A, & Enquist L.W. (2014). Characterization of a Replication-Incompetent Pseudorabies Virus Mutant Lacking the Sole Immediate Early Gene IE180. mBio, 5(6), e01850-14.

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NRF2 Protein Expression Analysis

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For western blot analysis of NRF2, 40 μg nuclear protein was mixed with Laemmli's buffer, incubated at 90°C for 10 min, and electrophoretically resolved using 10% SDS polyacrylamide gels and transblotted onto PVDF-Plus membrane (Micron Separations, Westboro, MA). A custom rabbit anti-Human NRF2 primary antibody (Cell Signaling Technology, Inc. Danvers, MA) was used to detect NRF2 with TFIIB as a loading control. Blots were then incubated with HRP conjugated secondary antibodies against rabbit IgG (Sigma-Aldrich, St Louis, MO). Protein-antibody complexes were detected using a chemiluminescent kit (Thermo Scientific, IL) with visualization using GeneMate blue autoradiography film (Bioexpress, Kaysville, UT).

Rudraiah S., Gu X., Hines R.N, & Manautou J.E. (2015). Oxidative Stress-responsive Transcription Factor NRF2 is Not Indispensable For The Human Hepatic Flavin-Containing Monooxygenase-3 (FMO3) Gene Expression in HepG2 Cells. Toxicology in vitro : an international journal published in association with BIBRA, 31, 54-59.

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