Chemiluminescence kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, China, Belgium

The Chemiluminescence kit is a laboratory product designed to detect and quantify specific molecules or analytes in a sample. It utilizes a chemiluminescent reaction, where a chemical reaction generates light that can be measured and used to determine the presence and concentration of the target analyte.

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Lab products found in correlation

Pvdf membrane, by Merck Group (17 mentions) Bca protein assay kit, by Thermo Fisher Scientific (9 mentions) Ripa lysis buffer, by Beyotime (8 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions) Gapdh, by Santa Cruz Biotechnology (6 mentions) Ripa buffer, by Merck Group (5 mentions) Protease inhibitor cocktail, by Merck Group (5 mentions) β actin, by Abcam (4 mentions) Lysis buffer, by Beyotime (4 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions) Pvdf membrane, by Bio-Rad (4 mentions) Chemidoc xr, by Bio-Rad (4 mentions) Cleaved caspase 3, by Cell Signaling Technology (3 mentions) Bca assay kit, by Merck Group (3 mentions) Chemidoc xrs imaging system, by Bio-Rad (3 mentions) Gapdh, by Abcam (3 mentions) Bca kit, by Beyotime (3 mentions) Polyvinylidene difluoride membrane, by Merck Group (3 mentions) Polyvinylidene fluoride membrane, by Merck Group (3 mentions) β actin, by Proteintech (3 mentions)

132 protocols using chemiluminescence kit

Western Blot Analysis of Renal Proteins

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Renal cortex tissue and cell lysates were lysed with 2% sodium dodecyl sulfate (SDS) solution with protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was measured with the bicinchoninic acid (BCA) reagent (Thermo Fisher Scientific). 100 μg proteins were loaded per lane and separated on SDS-polyacrylamide electrophoresis gel, and the proteins were then transferred onto polyvinylidene difluoride membrane. After blocking with 5% fat-free milk for 1 h, blots were incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Antigens were visualized with a chemiluminescence kit (Thermo Fisher Scientific). The protein bands were analyzed by Image J software (NIH) for densitometry.
2.14 Statistical analysisIn this study, qualitative data represents at least 3 separate experiments, and quantitative data is expressed as means ± SD. T-test was conducted to examine the statistical significance between 2 groups. ANOVA followed by Tukey's post-tests was used to determine the statistical differences among multiple groups. Statistical analysis was determined with the GraphPad Prism software. P < 0.05 was regarded as significant differences.

Wang Y., Zhu J., Liu Z., Shu S., Fu Y., Liu Y., Cai J., Tang C., Liu Y., Yin X, & Dong Z. (2020). The PINK1/PARK2/optineurin pathway of mitophagy is activated for protection in septic acute kidney injury. Redox Biology, 38, 101767.

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Western Blot Analysis of Transcriptional Regulators

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For Western blot, the total protein was extracted from Ea.hy926 cells receiving different treatments with radioimmunoprecipitation assay lysis (Beyotime, Shanghai, China) according to the recommended protocol (Li et al., 2020 (link)). Thirty micrograms of protein samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose filter membranes (Merck Millipore, Canada). After being blocked with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with anti-TBP antibody (1:2,000 dilution, YIFEIXUE BIO TECH, A0055), anti-Poly II antibody (1:2,000 dilution, Proteintech, 20655-I-AP), anti-BRG1 antibody (1:2,000 dilution, Abcam, Ab110641), and anti-c-Jun and anti-c-Fos antibody (1:500 dilution, Santa Cruz, sc-1694 or sc-52) overnight at 4°C. The Anti-Poly II antibody or anti-TBP antibody was used as a standard internal protein for normalizing. Horseradish peroxidase-conjugated immunoglobulin G (YIFEIXUE BIO TECH, China) was used to amplify the signal. After treatment with the chemiluminescence kit (Thermo Fisher Scientific, United States), protein signals were detected by ChemiDoc RXC + (Bio-Rad, United States).

Zhang Y., Wang H., Song M., Xu T., Chen X., Li T, & Wu T. (2020). Brahma-Related Gene 1 Deficiency in Endothelial Cells Ameliorates Vascular Inflammatory Responses in Mice. Frontiers in Cell and Developmental Biology, 8, 578790.

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Extracting and Analyzing Kidney Proteins

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Kidney tissue lysates were extracted with 2% SDS buffer as previously28 (link). Protein amounts were determined with the BCA kit from Thermo Scientific and equal amounts of protein were loaded in each lanes for SDS-polyacrylamide electrophoresis and then transferred to PVDF membrane. The blot was blocked with 5% milk for 1 hour at room temperature and then incubated with a specific primary antibody. The blot membrane was washed with TBST for 4 times and then incubated with horseradish peroxidase-conjugated secondary antibody, and signals were revealed using a chemiluminescence kit (Thermo Scientific).

Mei S., Li L., Wei Q., Hao J., Su Y., Mei C, & Dong Z. (2017). Double knockout of Bax and Bak from kidney proximal tubules reduces unilateral urethral obstruction associated apoptosis and renal interstitial fibrosis. Scientific Reports, 7, 44892.

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Western Blot Analysis of Kidney Tissue

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As described previously15 (link)33 (link)34 (link), the whole kidney tissue lysates were extracted with 2% SDS buffer. Protein amounts were determined with BCA reagent from Thermo Scientific. Equal amounts of protein (100 μg for kidney tissue lysates were loaded in each lane of SDS-polyacrylamide electrophoresis gel. Then resolved proteins were transferred to polyvinylidene difluoride membrane, which was then blocked with 5% bovine serum albumin for 1 hour at room temperature and further incubated overnight with a specific primary antibody. The blot membrane was washed with TBST for 4 times and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies. The blot signal was revealed with a chemiluminescence kit (Thermo Scientific).

Mei S., Livingston M., Hao J., li L., Mei C, & Dong Z. (2016). Autophagy is activated to protect against endotoxic acute kidney injury. Scientific Reports, 6, 22171.

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Western Blot Analysis of Apoptosis Markers

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Total cell lysates were collected and protein concentration was measured. Equal amount of proteins was separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% bovine serum albumin in TBST for 2h at room temperature and incubated with primary antibodies overnight at 4°C. Following the incubation with secondary antibodies at room temperature for 2h, proteins on the membrane were visualized with a chemiluminescence kit (Thermo Scientific). Primary antibodies are listed as follows: β-actin (1:1000, Cell Signaling Technology), FOXM1 (1:100, Santa Cruz Biotechnology), cleaved caspase-3 (1:1000, Cell Signaling Technology), cleaved caspase-7 (1:1000, Cell Signaling Technology), cleaved PARP (1:1000, Cell Signaling Technology) and ABCC10 (1:50, Santa Cruz Biotechnology).

Xie T., Geng J., Wang Y., Wang L., Huang M., Chen J., Zhang K., Xue L., Liu X., Mao X., Chen Y., Wang Q., Dai T., Ren L., Yu H., Wang R., Chen L., Chen C, & Chu X. (2016). FOXM1 evokes 5-fluorouracil resistance in colorectal cancer depending on ABCC10. Oncotarget, 8(5), 8574-8589.

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Comprehensive Protein Analysis Techniques

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SDS‐PAGE and western blot were conducted using the following antibodies: anti‐GAPDH (BM3874, Boster), anti‐TRIM55 (A15917, Abclonal), anti‐MMP2 (orb12416, Biorbyt), anti‐MMP9 (27306‐1‐AP, Proteintech), anti‐N‐Cadherin (22018‐1‐AP, Proteintech), anti‐Bak (A0498, Abclonal), anti‐Bax (sc‐7480), anti‐Bcl2 (ET7110‐51, HuaBio), anti‐Caspase 3 (ET1602‐39, HuaBio), anti‐c‐Myc (AF0358, Affinity), anti‐p21/Cip1 (27296‐1‐AP, Proteintech), anti‐E‐Cadherin (20874‐1‐AP, Proteintech), anti‐P27/Kip1 (NBP1‐32213, Novus), anti‐survivin (NBP2‐48494, Novus), mouse anti‐HA‐Tag mAb (AE065, Abclonal), and mouse anti His‐Tag mAb (AE003, Abclonal). Protein bands were developed with a chemiluminescence kit (ThermoFisher).

Lin M., Fang Z., Lin X., Zhou W., Wang Y., Han S., Ye M, & Zhu F. (2023). TRIM55 inhibits colorectal cancer development via enhancing protein degradation of c‐Myc. Cancer Medicine, 12(12), 13511-13521.

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Quantitative Protein Analysis by Western Blot

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Total protein was extracted using the SoniConvert™ system (DocSense) and RIPA buffer (Sigma-Aldrich; Merck KGaA). Total protein was denatured at 100˚C for 10 min and was quantitatively measured using a bicinchoninic acid assay kit (Sigma-Aldrich; Merck KGaA) in accordance with the manufacturer's protocol. Total protein (20 µg) were separated by 8-16% SDS-PAGE and transferred to nitrocellulose membranes. After blocking in 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) at room temperature for 30 min, the membranes were incubated with primary antibodies targeted against microtubule associated protein 1 light chain 3B (LC3B; cat. no. ab48394; 1:1,000; Abcam), p62 (cat. no. ab109012; 1:2,000; Abcam) and β-actin (cat. no. ab8226; 1:5,000; Abcam) at room temperature for 1 h. After three washes with PBS-T, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (cat. no. ab7090; 1:5,000; Abcam) at room temperature for 1 h. Protein bands were visualized using a chemiluminescence kit (Thermo Fisher Scientific, Inc.). Protein expression levels were quantified using Image J software (version 1.51; National Institutes of Health) with β-actin as the loading control.

Tang Q., Cao Y., Xiong W., Ke X., Zhang J., Xia Y, & Liu D. (2020). Glycyrrhizic acid exerts protective effects against hypoxia/reoxygenation-induced human coronary artery endothelial cell damage by regulating mitochondria. Experimental and Therapeutic Medicine, 20(1), 335-342.

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Western Blot Analysis of Protein Expression

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The IB procedure was performed according to the previously described protocol [31 (link)]. The antibodies utilized for IB analysis are specified in Supplementary Table 2. In brief, total cellular or tissues protein were extracted using RIPA buffer (R0278, Sigma-Aldrich) supplemented with PMSF protease inhibitor (36978, ThermoFisher Scientific), separated by SDS-PAGE gel electrophoresis and subsequently transferred onto a PVDF membrane (PB9220, ThermoFisher Scientific). The primary antibody was incubated and the secondary antibody was reacted, followed by detection of target bands using a chemiluminescence kit (89880, ThermoFisher Scientific).

Zhang D.Y., Zhu Y., Wu Q., Ma S., Ma Y., Shen Z.C., Wang Z., Sun W., Zhou Y.C., Wang D., Zhou S., Liu Z., Kwong L.N, & Lu Z. (2023). USP1 promotes cholangiocarcinoma progression by deubiquitinating PARP1 to prevent its proteasomal degradation. Cell Death & Disease, 14(10), 669.

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Protein Expression and Apoptosis Analysis

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The cells suspended in RIPA buffer (Sigma-Aldrich; Merck KGaA) and lysed using SoniConvert^® Sonicator (DocSense) and protein concentration was determined via BCA assay (Sigma-Aldrich; Merck KGaA) according to the manufacturer's instruction. In each lane, 20 µg total protein was applied to a 10% SDS-PAGE gel. Fractionated proteins were transferred onto a nitrocellulose membrane, and blocked using 5% BSA containing PBS at room temperature for 1 h. The membrane was incubated with the primary antibody specific to cleaved PARP, cleaved caspase-3 (1:1,000; cat. no. ab136812; Abcam), at 4°C overnight, then incubated with the secondary antibody goat anti-mouse IgG H&L antibody (HRP-labeled; 1:10,000; ab97040; Abcam) at 37°C for 1 h. Mouse anti-β-actin (1:5,000; cat. no. ab8226; Abcam) was used as an internal reference. Then blots were visualized with chemiluminescence kit (Thermo Scientific, Inc.) and quantitatively measured using Image J software (version-2.0; National Institutes of Health).

Sun Y., Deng R, & Zhang C. (2020). Erastin induces apoptotic and ferroptotic cell death by inducing ROS accumulation by causing mitochondrial dysfunction in gastric cancer cell HGC-27. Molecular Medicine Reports, 22(4), 2826-2832.

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Western Blot Analysis of Osteogenesis Markers

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Cells were washed twice with cold PBS and then lysed in lysis buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 2 mM dithiothreitol, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail) on ice for 15 minutes. Cell extracts were collected by centrifugation at 15,000 × g at 4°C for 30 minutes, run on 10% SDS-polyacrylamide gel electrophoresis gels, and transferred to polyvinylidene difluoride membranes by electroblotting. The membranes were blocked for 1 hour in a blocking buffer containing 5% powdered milk in Tris-buffered saline and Tween 20. They were then incubated overnight with a primary antibody at 4°C, followed by incubation with a secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Zhongshan Biotechnology Co., Beijing, China) for 30 minutes at room temperature, and visualized using a chemiluminescence kit (Thermo Scientific, Waltham, MA, USA). Specific antibodies against BMP2 (Abcam, Cambridge, UK, #ab14933), BMP4 (Abcam, #ab39973), RUNX2 (Abcam, #ab76956), SMURF2 (Abcam, #ab53316), TRAP (Abcam, #ab191406), MMP9 (Abcam, #ab38898), cathepsin K (Abcam, #ab187647), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-25778) were used.

Wang R., Feng Y., Xu H., Huang H., Zhao S., Wang Y., Li H., Cao J., Xu G, & Huang S. (2020). Synergistic effects of miR-708-5p and miR-708-3p accelerate the progression of osteoporosis. The Journal of International Medical Research, 48(12), 0300060520978015.

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