Immunoblotting was performed as previously described.15 (link) Briefly, proteins in cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF (polyvinylidene difluoride) membranes (Millipore, Bedford, MA, USA). The membranes were blocked by incubating with a Tris/saline/0.1% Tween-20 solution (TBS-T) containing 5% nonfat dry milk for 1 hour at room temperature and incubated with a primary antibody overnight at 4°C. Membranes were incubated with species-appropriate HRP-conjugated secondary antibodies for 1 hour and visualized using an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology), with detection by exposure to X-ray film (Kodak, Rochester, NY, USA) or Imagelab system (Bio-rad). Immunoprecipitation assays were performed as previously described.16 (link) Protein interactions and ubiquitination modifications were analyzed by incubating anti-antithrombin, anti-GFP, or anti-FLAG antibody with a Dynabeads Antibody Coupling Kit (Life Technologies, Invitrogen, CA) according to the manufacturer’s instructions, after which cell lysates were incubated with beads for 3h at room temperature. Precipitated proteins were washed three times with PBS containing 0.1% Tween-20 (PBS-T), eluted using Blue Loading Buffer Pack (Cell Signaling Technology), resolved by SDS-PAGE, and immunoblotted.
Enhanced chemiluminescence ecl kit
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Enhanced chemiluminescence (ECL) kit is a laboratory product designed to detect and quantify proteins in Western blot analysis. The kit utilizes a chemiluminescent substrate that emits light upon reaction with the enzyme horseradish peroxidase, enabling the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads antibody coupling kit, by Thermo Fisher Scientific (2 mentions)
Blue loading buffer pack, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Image lab system, by Bio-Rad (2 mentions)
X ray film, by Kodak (2 mentions)
Co ip assay kit, by Thermo Fisher Scientific (2 mentions)
Cell cycle detection kit, by Keygen Biotech (2 mentions)
Bicinchoninic acid bca protein assay reagent kit, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti mouse igg secondary antibody, by Merck Group (1 mentions)
Interleukin 2 (il 2), by Merck Group (1 mentions)
Il 10, by Merck Group (1 mentions)
Anti il 6, by Santa Cruz Biotechnology (1 mentions)
Secondary antibodies conjugated to horseradish peroxidase hrp, by Santa Cruz Biotechnology (1 mentions)
Aa 861, by Merck Group (1 mentions)
Anti il 2, by Santa Cruz Biotechnology (1 mentions)
Inf γ, by Merck Group (1 mentions)
Anti il 4, by Santa Cruz Biotechnology (1 mentions)
Anti il 10, by Santa Cruz Biotechnology (1 mentions)
Interleukin 6 (il 6), by Merck Group (1 mentions)
Interleukin 4 (il 4), by Merck Group (1 mentions)
12 protocols using enhanced chemiluminescence ecl kit
1
Protein Interaction and Modification Analysis
2
Optimized CEPO Dosage Administration Protocol
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Before administration of CEPO (Amgen, Thousand Oaks, CA, USA), the dose of CEPO was calculated and weighed in 50 µg/kg with reference of the total weight of rats in CEPO group. Then CEPO was dissolved in double distilled water until well mixed, and the concentration of CEPO was adjusted to 100 µg/ml; isoproterenol (ISO; 2 ml, 1 mg/bottle, Shanghai Harvest Pharmaceutical Co. Ltd., Shanghai, China); LY294002 (LY) (Selleck Chemicals, Houston, TX, USA); enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); Rabbit anti-rat pAkt (Ser473) and Akt monoclonal antibodies (cat. nos. 4058 and 4685 respectively; Cell Signaling Technology, Inc., Danvers, MA, USA); horseradish peroxidase labeled anti-rabbit IgG polyclonal antibody (cat. no. 7074; Cell Signaling Technology, Inc.). Bicinchoninic acid (BCA) protein assay reagent kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA).
3
Western Blotting Analysis of Chlamydial Proteins
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The whole protein of cells extracted by radioimmunoprecipitation assay (PIRA) buffer (Beyotime, China) was used for western blotting analysis according to the standard protocol. Equal amounts of protein were loaded and separated by 10% SDS-PAGE and transferred onto PVDF membranes. After locking with 5% skim milk for 2 h at room temperature, PVDF membranes were incubated overnight with our own primary antibodies. The primary antibody was probed using an HRP-conjugated goat anti-mouse IgG secondary antibody (Sigma-Aldrich) and visualized with using an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotech, Santa Cruz, CA, USA). Expression of the chlamydial plasmid proteins PGP3 and HSP60 was detected using GAPDH as a control. Three times Western Blotting data of proteins are shown in supplementary Fig. 4. As there are almost no miscellaneous bands, the representative original images are shown in supplementary Fig. 5.
4
Cytokine and Enzyme Inhibitor Assay
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Chemicals were of the purest analytical grade. Human recombinants IL-2, IL-4, IL-6, IL-10, INF-γ, and LIF, calpain substrate [N-Suc-Leu-Tyr-AMC (7-amido-4-methyl-coumarin)], AA861 (specific inhibitor of 5-LOX), and E64D (specific inhibitor of calpain) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mouse anti-cytochrome c antibody was from Cell Signalling Technology Inc. (Danvers, MA, USA); mouse anti-calpain-1 was from Calbiochem (Merck Darmstadt, Germany). Rabbit anti-LIF, anti-IL-2, anti-IL-4, anti-IL-6, anti-IL-10, anti-INF-γ, secondary antibodies conjugated to horseradish peroxidase (HRP), and enhanced chemiluminescence (ECL) kit were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Goat anti-rabbit conjugated to alkaline phosphatase (GAR-AP) was from Bio-Rad (Hercules, CA, USA).
5
Western Blot Analysis of Nrf2 and Antioxidant Proteins
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L-02 cells were homogenized in one volume of sample buffer (50 mM Tris-Cl, 100 mM DTT, 10% glycerol, and 2% sodium dodecyl sulfate (SDS)) and centrifuged at 14800 ×g at 4°C for 15 min to remove debris. The samples were subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with skim milk (5%), the blots were probed with primary antibodies (Abcam, Cambridge, UK) for Nrf2 (1 : 1000), HO-1 (1 : 1000), NQO1 (1 : 1000), GST (1 : 1000), and β-actin (1 : 1000) at 4°C for 8 h. Incubation with primary antibodies was followed by incubation with secondary antibodies (conjugated to horseradish peroxidase) after washing in Tris-buffered saline and Tween 20. The blots were processed using an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology Inc.) and exposed to film. All experiments were repeated three times.
6
Western Blotting for IκB-α Protein
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The cells were twice washed with cold PBS and collected by scraping after PRO-PREPTM (Intron Biotechnology, Seongnam, Korea). Then the cells were lysed in ice for 30 minutes with lysis buffer. The lysates were clarified by centrifugation in 12,000 rpm, 30 minutes at 4°C and the supernatant was collected. The protein concentration was determined using the Bradford assay. Then 30 μg of protein from each sample were boiled for 5 minutes and separated by SDS-PAGE using 12% separating gels. The protein was electrophoretically transferred to a Nitrocellulose Transfer Membrane (Whatman GmbH, Dassel, Germany) and blocked with 5% BSA in TBS-T buffer (20 mM Tris base, 1.37 mM NaCl, 0.05% Tween-20, pH 7.6) overnight at 4°C. The membrane was incubated with anti-IκB-α antibody (Santa Cruz Biotechnolgy, Santa Cruz, CA, USA) and antiphospho-IκB-α antibody (Santa Cruz Biotechnology) for 2 hours. After washing with TBS-T three times, the membrane was incubated with HRP-conjugated secondary antibody in TBS-T at 1:2,000 dilution for 1 hour. The blots were processed with an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology) and were performed with Bio-Imagin RAS 1000 plus (Fugifilm, Tokyo, Japan).
7
Protein Expression Analysis in Glioblastoma Cell Lines
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Harvested cells (U87 and U251) were lysed using RIPA (Beyotime Institute of Biotechnology) buffer on ice for 30 min and were centrifuged at 17,000 × g for 45 min at 4°C. The protein concentrations were measured by the BCA protein assay kit (Beyotime Institute of Biotechnology, Jiangsu, China). The proteins were processed by SDS-PAGE electrophoretically transferring to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked by Tween-Tris-buffered saline (TTBS) containing 5% non-fat milk for 2 h at room temperature and then incubated with primary antibodies as follows: USF1 (Santa Cruz Biotechnology), ALDH1A1 (Proteintech, USA), MMP-14 (Proteintech, USA), MMP-2 (Proteintech, USA), VE-cadherin (Abcam, UK), EphA2 (Abcam, UK), ERK (Abcam, UK), p-ERK (Abcam, UK), and GAPDH (Proteintech, USA) overnight at 4°C. After washing three times with TTBS, membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature and then developed with enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology) and scanned by ChemImager 5500 V2.03 software according to the manufacturer’s protocol. The relative integrated density values (IDVs) were calculated using Fluor Chen 2.0 software based on GAPDH as an internal control.
8
Western Blot Analysis of Cell Signaling
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Total protein was isolated using RIPA lysis (Trans-Gen Biotech) according to the manufacturer’s instructions. Approximately, an equal amount of protein was separated using 12% SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% non-fat milk for 1h at room temperature, the membranes were incubated with primary antibodies against CyclinE1 (ab33911, Abcam), Cleaved Caspase 3 (ab13847, Abcam), Runx2 (ab192256, Abcam), Osx (ab209484, Abcam), Ocn (ab133612, Abcam), SPTBN1 (ab72239, Abcam), TGF-β (ab215715, Abcam), p-STAT1 (ab109461, Abcam), p-SMAD3 (ab52903, Abcam), and β-actin (KM9001, Sungene Biotech, Tianjin, China). β-actin was served as the internal reference of Western Blot in this study. Subsequently, the membranes were incubated with HRP-labeled secondary antibody (1:5000) for 1 h. Finally, the detection of protein bands was performed using an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology, Dallas, TX, United States) according to the manufacturer’s instructions, and the bands of interest were visualized using a Bio-Rad imaging system (Bio-Rad Laboratories, Mississauga, ON, United States).
9
Investigating USP7-mediated Nrf2 Regulation
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P5091 was produced by Selleck (USA). Cell signaling technologies provided anti-USP7 and anti-bax antibodies (USA). Proteintech (China) produced anti-Keap1. Bioworld (Bioworld Technology, USA) provided anti-Nrf2 and anti-GAPDH antibodies. Antibodies produced against ubiquitin were bought from Life Technologies, as was the Co-IP assay kit (USA). Invitrogen produced the TRIzol reagent (USA). Takara Biomedical Technology (Beijing) Co., Ltd. provided the PrimeScript II 1st Strand cDNA Synthesis Kit. Keygen created the Cell Cycle Detection Kit (China). Santa Cruz Biotechnology provided the enhanced chemiluminescence (ECL) kit (USA). ROS Assay Kit was bought from Beyotime Biotechnology (China). Cusabio Technology (China) provided ELISA Kit. TUNEL Assay Kit was acquired from Roche (China). For immunoblot, immunofluorescence, and Co-IP assays, antibodies were diluted at 1:1000, 1:2500, 1:500, and 1:50, respectively.
10
Signaling Pathways in Fibroblast Regulation
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ML364 was manufactured by MedChemExpress (USA). Ang II was provided by Sigma‐Aldrich (USA). MG132 was a product of Enzo (USA). Anti‐USP2 and anti‐periostin antibodies were purchased from Proteintech (China). Anti‐collagen‐III antibody was manufactured by Abcam (United Kingdom). Anti‐CTGF and anti‐GAPDH antibodies were obtained from Boster (China) and Bioworld (Bioworld Technology, USA), respectively. Antibodies raised against ubiquitin, cyclin D1, P27, β‐catenin, GSK‐3β and P‐ GSK‐3β(Ser9) were provided by Cell Signaling Technology (USA). MTS assay reagents were manufactured by Promega (USA). The Co‐IP assay kit was purchased from Life Technologies (USA). Cell Cycle Detection Kit was manufactured by Keygen (China). The enhanced chemiluminescence (ECL) kit was provided by Santa Cruz Biotechnology (USA). Antibodies were diluted at 1:1000, 1:500 and 1:50 for immunoblot, immunofluorescence and Co‐IP assay, respectively.
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