The cells were harvested and lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with PMSF (1 mM) and protease inhibitor cocktail (Cell Signaling Technology, Massachusetts, USA). The protein concentration was determined using Bradford protein assay (Sigma, Missouri, USA). The total protein was then resolved on SDS-PAGE (8–12%) and transferred onto PVDF membrane (Millipore, Massachusetts, USA). After the transfer, using protein ladder as reference, membranes were cut according to the protein of interest (as shown in the results). The further western blot protocol was followed as standard procedure [17 (link)] and e nhanced chemiluminescence (ECL) substrate (Bio-Rad, California, USA) was used to develop the blots. Documentation of the blots was done using chemidoc system (Azure Biosystems 600, California, USA).
Enhanced chemiluminescence ecl substrate
Manufactured by Bio-Rad
Sourced in United States
Enhanced chemiluminescence (ECL) substrate is a laboratory reagent used for the detection of proteins in Western blot analysis. It is a light-emitting chemical reaction that occurs when the substrate is exposed to a specific enzyme, resulting in the emission of light. The intensity of the light signal is proportional to the amount of the target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Bradford protein assay, by Merck Group (1 mentions)
Xanthan gum, by Merck Group (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Bio-Rad (1 mentions)
Linoleic acid, by Merck Group (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
Trypsin, by Lonza (1 mentions)
Radioimmunoprecipitation assay ripa buffer, by Cell Signaling Technology (1 mentions)
Ponceau s, by Boston BioProducts (1 mentions)
Cellytic m buffer, by Merck Group (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Ab30647, by Abcam (1 mentions)
11 protocols using enhanced chemiluminescence ecl substrate
1
Western Blot Protein Analysis Protocol
2
Optimizing Leptin Signaling Assays
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All the chemicals, unless specified, including xanthan gum, linoleic acid and anti-mouse secondary antibody, were purchased from Sigma (St. Quentin Fallavier, France). Fura-2/AM was purchased from Life Technologies (Bordeaux, France). Trypsin, RPMI 1640 medium and fetal calf serum (FCS) were bought from Lonza Verviers (Belgium). Pegylated mouse leptin antagonist (mutant L39A/D40A/F41A) was purchased from CliniSciences (Montluçon, France). Accell siRNA for leptin, leptin receptor (Ob-Rb) and non-targeting (labeled with DY-547) were procured from Dharmacon™ (USA). The ELISA kit for leptin (A05176) was obtained from Bertin Technologies (Aix-en-Provence, France). Alexa 568 donkey anti-rabbit (A10042), Alexa 488 green goat anti-rabbit (A11034) and other molecular biology reagents including TRIzol were from Invitrogen (Waltham, MA, USA). The rabbit anti-CD36 (HPA002018) antibody was bought from Atlas (Cambridge, UK). The anti-β-actin antibodies were from Santa Cruz Biotechnology (Dallas, Texas, USA). The anti-leptin (GTX17629) and anti-Ob-Rb (GTX27211) antibodies were bought from GeneTex (Irvine, CA, USA). The anti-rabbit (7074S) goat antibodies were bought from Cell signaling (Charles-Renard, France). The polyvinylidene fluoride (PVDF) membranes and enhanced chemiluminescence (ECL) substrate were procured from Bio-Rad (Marnes-la-Coquette, France).
3
Western Blot Analysis of Skeletal Muscle and Neural Tissues
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Tissue was taken from adult mice and extracted using Radioimmunoprecipitation Assay buffer (RIPA; Cell Signaling Technology, Danvers, MA, USA). Protein concentrations were determined in accordance with the standard Bradford Assay using bovine serum albumin (BSA) standards. Protein samples (10μg/lane skeletal muscle; 40μg/lane spinal cord, brain, and sciatic nerve; 5n g/lane rhMG53) were separated by SDS-PAGE at room temperature on 10% gels at 150 V and were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Blots were stained with Ponceau S (Boston Bioproducts, Ashland, MA, USA) to visualize total protein. Blots were probed for TRIM72/MG53 with a custom polyclonal antibody (Pacific Immunology, San Diego, CA, USA), and anti-rabbit horseradish peroxidase (HRP)–conjugated secondary antibodies (Cell Signaling Technology). The blots were developed using enhanced chemiluminescence (ECL) substrate (Bio-Rad). An Azure Biosystems imager was used to visualize chemiluminescent blots.
4
Western Blot Analysis of Signaling Proteins
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Cells were lysed using CelLytic M buffer (Sigma-Aldrich) containing a 1 × protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Beverly, MA, USA). The protein concentration was assessed using Bradford reagent (Sigma-Aldrich). Equal amounts of protein (50 µg) were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels, and transferred onto a nitrocellulose membranes (Amersham Biosciences, Uppsala, Sweden). After blocking with 5% skimmed milk, the membranes were incubated with rabbit phospho-STAT6, rabbit phospho-ERK, rabbit phospho-P38, rabbit phospho-JNK, rabbit phospho-AKT, rabbit FOXA2 (Cell Signaling Technology), rabbit SPDEF (Abcam, Cambridge, UK) and mouse MUC5AC antibodies (Merck Millipore, Darmstadt, Germany), overnight at 4 °C. Then, these were further incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (GeneTex, Irvine, CA, USA). For band detection, an enhanced chemiluminescence (ECL) substrate (Bio-Rad Laboratories, Hercules, CA, USA) was used. The band density was quantified using ImageJ software (NIH).
5
Quantification of Mucosal Immune Proteins
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Total protein from mouse ilea was prepared using a total protein extraction kit (Applygen, Beijing, China) according to the manufacturer's instructions. Protein samples were resolved by SDS-PAGE on pre-cast 4–15% gels (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA) and incubated overnight at 4 °C with rabbit polyclonal antibodies against Foxp3 (ab10901), RORγt (ab207082), STAT3 (ab68153), p-STAT3 (phospho S727, ab30647), STAT5 (ab16276), p-STAT5 (phospho Y694, ab32364), and β-actin (ab179467, Abcam, Cambridge, UK). Horseradish peroxidase-conjugated anti-rabbit polyclonal antibodies (Goat anti-rabbit IgG-HRP, ab6721, Abcam) were used as secondary antibodies and detected using enhanced chemiluminescence (ECL) substrate (Bio-Rad). Band densitometry was performed using Image Lab software (Bio-Rad). The relative index was represented as the ratio of the selected protein/β-actin, and was the average of three biological replicates.
6
Quantitative Protein Analysis via RIPA-ROCK1
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Radioimmunoprecipitation assay (RIPA) containing protease inhibitor was applied for extraction of total protein. 50 μg proteins of each sample were electrophoresed on 12% TruPAGE™ Precast Gels using Sigma-Aldrich® Dual Run and Blot System (Sigma) and transferred to Immobilon-E Polyvinylidene Fluoride Membrane (Sigma) through the Trans-Blot Turbo Transfer System (Bio-Rad). The non-specific proteins were blocked and the membranes were incubated with primary antibodies of ROCK1 (Abcam, Cambridge, UK; ab97592, 1:1000) or GAPDH (Abcam, ab128915, 1:1000) at 4 °C overnight. After the incubation of Goat Anti-Rabbit IgG H&L secondary antibody (ab205718, 1:3000), immunoreactive blots were visualized via Enhanced Chemiluminescence (ECL) Substrate (Bio-Rad). GAPDH were used as the internal reference, and the protein intensity was analyzed by ImageJ software (NIH).
7
Murine Macrophage Cell Line Protocol
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Water containers were purchased from VWR (Alfragide, Portugal). Raw 264.7 cell line was acquired from American Type Culture Collection (ATCC-TIB-71) and kindly supplied by Dr. Otília Vieira (Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal). Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Massachusetts, USA). LPS from Escherichia coli (serotype 026:B6) was obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). The protease and phosphatase inhibitor cocktails were obtained from Roche (Mannheim, Germany). Acrylamide was purchased from Promega (Madison, WI, USA) and the Polyvinylidene difluoride (PVDF) membranes were from Millipore Corp. (Bedford, MA, USA). The primary antibody mouse anti-iNOS was from R&D Systems (Minneapolis, MN, USA) and mouse anti-β-Tubulin was obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). The anti-mouse Horseradish Peroxidase conjugated secondary antibody was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Enhanced chemiluminescence (ECL) substrate was obtained from Bio-Rad (Hercules, USA). All other reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA).
8
Western Blot Analysis of PAX6 and GAPDH
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Cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma) containing the protease inhibitor cocktail (Sigma) to isolate total protein, and the proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. Following blocking with 5% skim milk for 2 h, the membranes were incubated with anti-PAX6 (1:1000, ab195045, Abcam) and anti-GAPDH antibodies (1:2000, ab8245, Abcam) at 4°C overnight. After washing, the membranes were incubated with the secondary antibody (Abcam) conjugated with horseradish peroxidase for 2 h at room temperature. Enhanced chemiluminescence (ECL) substrate (Bio-Rad) was used to visualize the protein bands [22 (link)].
9
Protein Extraction and Quantification from Varicose Veins
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Proteins were extracted from 31 varicose, and 34 control veins (100 µg) using RIPA buffer with protease inhibitor cocktails described earlier [6 (link)]. Protein concentrations were determined with the Bradford reagent as per manufactures instructions (Bio-Rad, Hercules, CA, USA). Total proteins were resolved on a 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% BSA in TBS for 1 h to reduce the non-specific binding of proteins, and membranes were incubated with primary antibodies (Supplementary Table S2 ) at 4 °C overnight. After rinsing thrice with TBS containing 0.1% Tween-20 (TBST), the membrane was incubated with HRP-conjugated secondary antibodies for 1 h at 37 °C in TBST. The chemiluminescence signal was detected using enhanced chemiluminescence (ECL) substrate (Bio-Rad, USA), and chemiluminescence measurements were performed using the Chemi Doc imaging system. Image J software was used for densitometry of immunoreactive bands.
10
Western Blot Analysis of Signaling Pathways
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Cells were lysed in ice-cold lysis buffer [50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol], phosphatase and protease inhibitors (Roche). Equal protein amount (20 µg) from each cell lysate was separated on sodium dodecyl sulfate (SDS) 10% polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked with 3% bovine serum albumin (BSA) for 1 h and incubated with antibodies against IκBα (9242), p-p38 (Thr180/Tyr182) (9211), total p38 (9212), p-ERK1/2 (Thr202/Tyr204) (9101), and total ERK 1/2 (9102) from Cell Signaling and β-actin (sc-47778) from Santa Cruz Biotechnology. The membranes were then incubated with the respective horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h and developed using enhanced chemiluminescence (ECL) substrate (BioRad).
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