SW1353 cells (5x105 in each dish) were seeded in a 6 cm dish and treated with 10 ng/ml IL-1β and/or 20 mM irisin at 37˚C for 24 h, then RIPA lysis buffer (Cell Signaling Technology, Inc.) was used to extract protein. Protein concentration was determined using a bicinchoninic acid kit (Sigma-Aldrich; Merck KGaA). A total of 20 µg of protein was loaded into each well of a 10% SDS-PAGE gel. After gel electrophoresis, the protein bands were separated from the gel, transferred to PVDF membranes after blocking with 5% non-fat milk at room temperature for 1 h by transfer electrophoresis and then incubated at 37˚C for 1 h with the following antibodies: MMP-13 (1:10,000; cat. no. DF6494; Affinity Biosciences), Col II (1:10,000 dilution; cat. no. AF5456; Affinity Biosciences), phospho-NFκB (1:10,000; cat. no. AF2006; Affinity Biosciences), inhibitor of NF-κB (IκB) α (1:10,000; cat. no. AF002; Affinity Biosciences), Wnt-1 (1:10,000; cat. no. DF514; Affinity Biosciences) and β-catenin (1:2,000; cat. no. ab1008; Abcam). β-actin (cat. no. ab051, Abcam) was used as the endogenous control. The membranes were then incubated with a goat anti-rabbit secondary antibody (1:10,000; cat. no. ab97051; Abcam) at 37˚C for 1 h. Protein bands were visualized with an ECL kit (Abcam). The data of study groups were quantitatively analyzed relative to the NC group using SPSS 19.0 (IBM Corp.).
Ecl kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The ECL kit is a lab equipment product that enables chemiluminescent detection of proteins or nucleic acids. It provides the necessary reagents for performing Western blot analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Merck Group (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Bromophenol blue, by Merck Group (3 mentions)
Immobilon p membrane, by Merck Group (3 mentions)
Hrp coupled secondary antibodies, by Promega (3 mentions)
Caspase 3, by Abcam (3 mentions)
Pvdf membrane, by Pall Corporation (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Genegnome machine, by Syngene (2 mentions)
P akt, by Abcam (2 mentions)
P erk, by Abcam (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Sds polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Bcl 2, by Abcam (2 mentions)
β mercaptoethanol, by Avantor (2 mentions)
β actin, by Abcam (2 mentions)
Ab200834, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
36 protocols using ecl kit
1
Irisin Modulates Inflammation and ECM Proteins
2
Insulin and Glucose Regulation Proteins
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The expressions of insulin, GLUT2, PGC-1α, GCK, and G6Pase were determined by western blot and GAPDH was employed as control. Pancreas was lysed using cell lysis reagent (Sigma) and phosphatase inhibitors (Sigma) and lysed by Tissue Homogenizer (Bertin Technologies, France). The crude lysate was transferred to new Eppendorf tubes. Each sample was added to 20 μL 2x sample loading buffer (0.125 M of 5 M Tris-HCl, Amresco; 20% glycerol, Usb; 4% of 10% sodium dodecyl sulfate, Amresco; 1% β-mercaptoethanol, Amresco; 0.2% of 0.05% (w/v) bromophenol blue, Sigma) and boiled for 5 min before loading. Proteins were separated by SDS-PAGE, transferred to immobilon P membrane (Millipore), and probed with different antibodies as indicated. All antibodies were purchased from Cell Signaling (Beverly, MA). The results were visualized using ECL kit (Abcam) and observed by GeneGnome machine (Syngene).
3
Optimized Western Blot Protocol
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Dulbecco’s modified Eagle’s medium (DMEM) and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, Missouri, USA). PVDF membranes were purchased from Pall Corporation (Ann Arbor, MI, USA). Actin and Cox-2 antibodies were purchased from Abcam (Cambridge, MA, USA). HRP-coupled secondary antibodies were purchased from Promega Corporation (Madison, WI, USA). The ECL kit was purchased from Abcam (Milton, Cambridge, UK). Fetal bovine serum (FBS), penicillin-streptomycin, glycine, lysis buffer solution, phosphate buffer saline (PBS), bovine serum albumin (BSA), tris-buffered saline with Tween 20® (TBST), HEPES buffer, sodium dodecyl sulfate (SDS), well plates, pentane, diethyl ether, hexane, ethyl acetate, anisaldehyde, ethanol, paraformaldehyde, trypan blue, Ficoll (Histopaque-1077). Crystal violet, trypsin, d6-DMSO and CDCl3 were purchased from Sigma (St. Louis, Missouri, USA) unless otherwise stated.
4
Hippocampal Protein Expression Analysis
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The rats were sacrificed under deep anaesthesia (2.5 g/kg urethane). The hippocampal tissues were isolated. After extracting the total protein, the concentration was determined using the Bradford method. The proteins were subjected to SDS-PAGE with 12% precast gels and then transferred to PVDF membranes, which were blocked for 1 h in 5% non fat dry milk in TBS-T. The following primary antibodies were used at the indicated dilutions: Nrf2 (1:500; Abcam; ab31163; USA), HO-1 (1:200; Abcam; ab13243; USA), and NQO1 (1:200; Abcam; ab28947; USA). The membranes were then probed with HRP-conjugated secondary antibodies for 1 h. The signals were detected using an ECL kit (Abcam; USA) according to the manufacturer’s instructions. The changes in the relative protein expression are presented as the ratio of the integrated optical density of the target protein bands to that of the β-actin protein band.
5
Western Blot Analysis of Protein Extraction
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Total protein from 16HBE cells was extracted using RIPA lysis buffer (Elabscience, Wuhan, China). After protein concentrations were determined by BCA Protein Assay Kit (Takara, Japan), equal amount of protein for each sample was resolved with SDS-PAGE (10%) and then immobilized onto PVDF membranes (Roche, Basel, Switzerland). The membranes were soaked in 5% non-fat dried milk in PBS and incubated with antibodies against MUC5AC (1:1,000, ab24071, Abcam), p65 (0.5 µg/mL, ab16502, Abcam), Lamin B1 (0.1 µg/mL, ab16048, Abcam), and β-actin (1:5,000; ab8226, Abcam) overnight at 4 °C respectively, and then incubation with corresponding secondary antibody at room temperature for 60 min. The blots were revealed with an ECL kit (Abcam) and analyzed by Image Software (NIH, Bethesda, MD, USA).
6
Protein Expression Analysis of Kidney Tissues
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The kidney tissues were lysed in RIPA buffer, lysates were resolved, and we conducted electrophoresis in SDS-polyacrylamide gels. Proteins on SDS-Page were subsequently transferred onto nitrocellulose membranes. Membranes were incubated in blocking buffer at 22°C for 1 h, and with primary antibody (anti-WT1, ZO-1, AMPK, NOX-4, Abcam, Shanghai, China) diluted in blocking buffer at 4°C overnight, followed by another 1-h secondary HRP antibody (anti-rabbit, Abcam, Shanghai, China) incubation at 22°C after washing with TBS-Tween. Enhanced chemiluminescence detection was performed using the ECL Kit (Abcam, Shanghai, China).
7
Investigating Ras-Induced Cell Signaling
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HaCaT-ras II4 cells were treated with two different concentrations of F2 (25 and 50 μg/mL) for 48 h. Adherent and non-adherent cells were collected on ice, washed twice with PBS, lysed with lysis buffer and centrifuged at 12,000 g for 10 min at 4 °C. The cell lysate was heated at 100 °C for 5 min, and the protein content was determined by the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA).
Equal protein concentrations were subjected to Western blot analysis as described previously [20 (link)]. The PVDF membranes were blocked with blocking buffer (1× TBS, 0.1% Tween-20, 5% skim milk) for 2 h and then probed with primary rabbit polyclonal antibodies to Caspase-3, AKT, p-AKT, ERK and p-ERK (Abcam, Cambridge, UK) and mouse monoclonal antibodies to actin, p53, p21, Bcl-2, BAX (Santa Cruz, CA) at dilution ranging from 1/1000 – 1/5000 at 4 °C overnight. Later, the primary antibodies were washed away with TBST for 2 h and the membranes were treated with horseradish peroxidase (HRP)-coupled secondary antibodies in blocking buffer at a dilution of 1/5000 (Abcam, Cambridge, UK) for 1 h, and washed with TBST afterwards. Finally, detection of each protein was performed using the chemiluminescence ECL kit (Abcam plc, Cambridge, UK). Blot images were obtained with the image lab Software (BioRad, Chemidoc imaging instrument).
Equal protein concentrations were subjected to Western blot analysis as described previously [20 (link)]. The PVDF membranes were blocked with blocking buffer (1× TBS, 0.1% Tween-20, 5% skim milk) for 2 h and then probed with primary rabbit polyclonal antibodies to Caspase-3, AKT, p-AKT, ERK and p-ERK (Abcam, Cambridge, UK) and mouse monoclonal antibodies to actin, p53, p21, Bcl-2, BAX (Santa Cruz, CA) at dilution ranging from 1/1000 – 1/5000 at 4 °C overnight. Later, the primary antibodies were washed away with TBST for 2 h and the membranes were treated with horseradish peroxidase (HRP)-coupled secondary antibodies in blocking buffer at a dilution of 1/5000 (Abcam, Cambridge, UK) for 1 h, and washed with TBST afterwards. Finally, detection of each protein was performed using the chemiluminescence ECL kit (Abcam plc, Cambridge, UK). Blot images were obtained with the image lab Software (BioRad, Chemidoc imaging instrument).
8
Investigating Apoptosis Pathways in MDA-MB-231 Cells
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MDA-MB-231 cells were treated with different concentrations of F1 and F2 (25 and 50 μg/ml) for 48 h. The adherent and non-adherent MDA-MB-231 cells were collected on ice, washed twice with PBS, lysed with lysis buffer, and centrifuged at 12,000 g for 10 min at 4°C. The cell lysate was heated at 100°C for 5 min, and the protein content was determined by the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). The same amount of proteins was loaded to a 10% SDS-PAGE. Proteins were then transferred to PVDF membrane (Pall Corporation, Ann Arbor, USA) and blocked with 5% skim milk for 2 h. The membranes were probed with primary antibodies against Actin, p53, Bcl-2, Bax, Caspase-3, PARP, Akt, p-Akt, Erk, and p-Erk (Abcam, Cambridge, USA) at 4°C overnight. Later, the primary antibodies were washed away with TBST for 1 h and the membranes were treated with HRP-coupled secondary antibodies (Promega Corp., Madison, USA) for 1 h, and washed with TBST afterwards. Finally, Detection of each protein was performed using the ECL kit (Abcam plc, 330 Cambridge Science Park, Cambridge UK).
9
Western Blot Analysis of Cell Lysates
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10
Protein Expression Analysis by Western Blot
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The expressions of p65, IкBα, p-IкBα and GAP43 were determined by western blotting and β-actin was employed as control while HDAC1 was used as a control of p65 in nucleus. Cell extracts were lysed in RIPA buffer (Beyotime, China) containing Complete Protease Inhibitor Cocktail and 2 mM PMSF. About 20 μg of total protein was separated by 10% SDS-PAGE and transferred to 0.45 μm Nitrocellulose Membrane (Millipore, USA). Protein concentrations were determined by BCA Protein Assay Kit (Beyotime). The membranes were incubated with primary antibodies at 4°C overnight and then hybridized with appropriate HRP-conjugated secondary antibody (Amersham Pharmacia Biotech) at room temperature for 2 h. The results were visualized using ECL kit (abcam) and observed by GeneGnome mechine (Syngene).
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