Western blot analysis was performed as described previously.33 (link) After washing cells twice in ice-cold PBS, protein extracts were prepared by incubating cell pellets in JS buffer (50 mM HEPES [pH 7.5] containing 150 mM NaCl, 1% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 5 mM EGTA, 1 mM Na3VO4, and 1X protease inhibitor cocktail). Protein concentrations were determined with Bio-Rad Protein Assay, and equal amounts of proteins were separated by SDS-PAGE (10% polyacrylamide gel). The separated proteins were transferred to nitrocellulose membranes (GE Healthcare, Milan, Italy). Membranes were blocked for 1 h with 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween-20. Primary antibodies were incubated at 4°C overnight, and peroxidase-conjugated secondary antibodies were used to perform an enhanced chemiluminescence (ECL Star, Euroclone, Milan, Italy) reaction, according to the manufacturer’s protocol, in order to identify target proteins. Primary antibodies used were as follows: anti-β3-tubulin, anti-Sox2, anti-GFAP, anti-Nanog, and anti-β-actin (Sigma-Aldrich).
Ecl star
Manufactured by Euroclone
Sourced in Italy
ECL Star is a chemiluminescence detection system for Western blot analysis. It provides sensitive and quantitative detection of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Hybond p, by Merck Group (2 mentions)
Pageruler, by Thermo Fisher Scientific (2 mentions)
Chemidoc gel imaging system, by Bio-Rad (2 mentions)
Protein assay, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Anti sox2, by Merck Group (1 mentions)
Anti gfap, by Merck Group (1 mentions)
Anti β3 tubulin, by Merck Group (1 mentions)
Anti nanog, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Anti gapdh g 9, by Santa Cruz Biotechnology (1 mentions)
Chemidoc image acquisition and analysis tool, by Bio-Rad (1 mentions)
Anti sirt1 d1d7, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cytiva (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (1 mentions)
Anti gapdh sc 25778, by Santa Cruz Biotechnology (1 mentions)
Complete mini protease and phosphatase inhibitor cocktails, by Roche (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
8 protocols using ecl star
1
Protein Expression Analysis by Western Blot
2
Western Blot Analysis of EGF and His-tag
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Protein samples were dissolved in the loading buffer for few minutes and then loaded, without prior heating, on a 10% (w/v) reducing SDS‐PAGE that was run at 20 V/cm for 60 min. After electrophoresis, the protein bands were visualized by Coomassie Blue staining and photographed or transferred onto a PVDF membrane.
After blotting, the membrane was blocked using 5% (w/v) non‐fat dried milk in PBS solution containing 0.1% (v/v) Tween 20. Incubation was carried on in the same buffer with an anti‐human EGF polyclonal antibody (PeproTech) diluted 1:1000 for 16 h, and subsequently with secondary horseradish peroxidase‐conjugated antibody (Sigma‐Aldrich, #A6154) diluted 1:10,000 for 1 h. For His‐tag detection, a primary mAb (GE Healthcare, #274710‐01) was applied in the same conditions and it was recognized by a secondary anti‐mouse peroxidase‐conjugated antibody. Immunodetection was performed using the chemo‐luminescent substrate (ECLStar, EuroClone) according to the manufacturer's instructions.
After blotting, the membrane was blocked using 5% (w/v) non‐fat dried milk in PBS solution containing 0.1% (v/v) Tween 20. Incubation was carried on in the same buffer with an anti‐human EGF polyclonal antibody (PeproTech) diluted 1:1000 for 16 h, and subsequently with secondary horseradish peroxidase‐conjugated antibody (Sigma‐Aldrich, #A6154) diluted 1:10,000 for 1 h. For His‐tag detection, a primary mAb (GE Healthcare, #274710‐01) was applied in the same conditions and it was recognized by a secondary anti‐mouse peroxidase‐conjugated antibody. Immunodetection was performed using the chemo‐luminescent substrate (ECLStar, EuroClone) according to the manufacturer's instructions.
3
Quantification of SIRT1 Protein Levels
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Total proteins were extracted from the aortic and heart tissues using a RIPA buffer with protease inhibitors, and quantified using the Bradford protein assay.
A total of 45 μg protein per well was separated via electrophoresis on 4–15% sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were then blocked with 5% milk TBST buffer (TBS plus 0.1% Tween-20) for 1 h at room temperature, and incubated with the primary antibody, anti-SIRT1 (D1D7) (Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-GAPDH (G-9) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4 °C, washed three times with the TBST buffer and incubated with the corresponding secondary antibodies at room temperature for 45 min, anti-rabbit and anti-mouse IgG heavy and light chain (Bethyl Laboratories, Inc., Montgomery, TX, USA), respectively. The signals were detected using the “Enhanced Chemiluminescent Substrate” method with ECL Star (EuroClone S.p.a, Milan, Italy) and analyzed with the ImageLab software, using the Chemidoc image acquisition and analysis tool (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All data were expressed as the mean ± SD.
A total of 45 μg protein per well was separated via electrophoresis on 4–15% sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were then blocked with 5% milk TBST buffer (TBS plus 0.1% Tween-20) for 1 h at room temperature, and incubated with the primary antibody, anti-SIRT1 (D1D7) (Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-GAPDH (G-9) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4 °C, washed three times with the TBST buffer and incubated with the corresponding secondary antibodies at room temperature for 45 min, anti-rabbit and anti-mouse IgG heavy and light chain (Bethyl Laboratories, Inc., Montgomery, TX, USA), respectively. The signals were detected using the “Enhanced Chemiluminescent Substrate” method with ECL Star (EuroClone S.p.a, Milan, Italy) and analyzed with the ImageLab software, using the Chemidoc image acquisition and analysis tool (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All data were expressed as the mean ± SD.
4
Western Blot Analysis of DNA Damage Signaling
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Cell pellets were resuspended in Laemmli buffer [62.5 mM Tris-HCl (pH 6.8), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue], sonicated, denatured for 5 min and separated on SDS-PAGE. The proteins were then transferred to nitrocellulose membranes and blocked with 5% non-fat dry milk or with 5% Bovine Serum Albumin (Sigma-Aldrich) in Tris-Buffered Saline with 0.1% Tween 20 (Sigma-Aldrich).
Primary antibodies used were: polyclonal anti-phosphoChk1 (Ser345), monoclonal anti-Chk1, polyclonal anti-phosphoChk2 (Thr68) and monoclonal anti-Chk2 from Cell Signaling; polyclonal anti-p21, monoclonal anti-p53, polyclonal anti-phosphoCdc25C (Ser216) and monoclonal Cdc25C from Santa Cruz Biotechnology; monoclonal anti-GFP (Covance); monoclonal anti-Actin (Sigma-Aldrich). Appropriate horseradish peroxidase conjugated secondary antibodies (Amersham Biosciences) were added and proteins were then detected using the enhanced chemiluminescence reagent ECL Star (Euroclone).
Primary antibodies used were: polyclonal anti-phosphoChk1 (Ser345), monoclonal anti-Chk1, polyclonal anti-phosphoChk2 (Thr68) and monoclonal anti-Chk2 from Cell Signaling; polyclonal anti-p21, monoclonal anti-p53, polyclonal anti-phosphoCdc25C (Ser216) and monoclonal Cdc25C from Santa Cruz Biotechnology; monoclonal anti-GFP (Covance); monoclonal anti-Actin (Sigma-Aldrich). Appropriate horseradish peroxidase conjugated secondary antibodies (Amersham Biosciences) were added and proteins were then detected using the enhanced chemiluminescence reagent ECL Star (Euroclone).
5
Western Blot Analysis of Bacterial Proteins
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Bacteria pellets were resuspended in phosphate-buffered saline (PBS) with 1× final sample buffer (FSB) and, after boiling at 100°C, loaded on 12.5% SDS-PAGE. A protein molecular weight marker (page ruler; Thermo Fisher) was included in each electrophoresis run. Proteins were transferred onto nitrocellulose membranes (Hybond-P; Millipore) and filter incubated with mouse monoclonal anti-FLAG M2 antibody (Sigma-Aldrich), rabbit polyclonal anti-OmpA, or anti-VirF halon (68 (link)) antibodies. Secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit IgG (Sigma-Aldrich). Signals were produced with ECL Star (Euroclone) and detected with ChemiDoc gel imaging system (Bio-Rad Laboratories). The densitometric analysis was performed by ImageJ software.
6
Western Blot Analysis of Bacterial Proteins
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Bacteria pellets were resuspended in PBS with 1X Final Sample Buffer (FSB) and, after boiling at 100 °C, loaded on 12.5% SDS-PAGE. A protein molecular weight marker (Page ruler; Thermo Fisher) was included in each electrophoresis run. Proteins were transferred to nitrocellulose membranes (Hybond-P, Millipore), and the immunoblotting was carried out with Monoclonal ANTI-FLAG® M2 antibody (Sigma-Aldrich) and rabbit polyclonal anti-OmpA53 (link),54 (link) as primary antibodies, and HRP conjugated goat anti-mouse and anti-rabbit IgG antibody as the secondary antibodies (Sigma-Aldrich). Signals were produced with ECL Star (Euroclone) and detected with ChemiDoc™ Gel Imaging System (Bio-Rad Laboratories). The densitometric analysis was performed by ImageJ software55 (link).
7
Western Blot Analysis of FLAG-tagged Proteins
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Cells were lysed with RIPA buffer (Sigma) supplemented with Complete Mini protease and phosphatase inhibitor cocktails (Roche). 5 μg of proteins were loaded on NuPAGE 4–12% Bis-Tris gel and transferred on Nitrocellulose membranes with iBlot system (Life Technologies).
After blocking in 5% milk-TBST, membranes were incubated overnight with primary antibodies anti-FLAG (M2, Sigma RRID: AB_259529) and anti-GAPDH (sc-25778, Santa Cruz Biotechnology RRID : AB_10167668).
After 1 hour incubation in the appropriate HRP conjugated secondary antibodies (Merck Millipore), detection was performed with ECL Star (Euroclone) with Azure Biosystem C400 camera. Densitometric quantification was performed with FIJI (RRID : SCR_002285) (26 (link)).
After blocking in 5% milk-TBST, membranes were incubated overnight with primary antibodies anti-FLAG (M2, Sigma RRID: AB_259529) and anti-GAPDH (sc-25778, Santa Cruz Biotechnology RRID : AB_10167668).
After 1 hour incubation in the appropriate HRP conjugated secondary antibodies (Merck Millipore), detection was performed with ECL Star (Euroclone) with Azure Biosystem C400 camera. Densitometric quantification was performed with FIJI (RRID : SCR_002285) (26 (link)).
8
Cell Lysis and Immunoprecipitation Protocol
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Cells were lysed in saline buffer 1% v/v Triton (NaCl, 150 mM; Tris-HCl, 50 mM, pH8; EDTA, 5 mM) or, for immunoprecipitation assay, in saline buffer 0.5% NP40 (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 100 mM EDTA,0.5% NP40) containing aprotinin (5 μg/ml), leupeptin (10 μg/ml), pepstatin (2 μg/ml), 0.5 mm PMSF, 2 mM orthovanadate, and 10 mM NaF. Lysates were clarified by centrifugation at 18.000 rcf for 10 min and subjected to immunoprecipitation with the indicated antibody at the indicated dilution. The precipitate samples and aliquots of whole cell lysates (50 μg) were resolved on sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred on nitrocellulose membrane for 7 min with a Transblot turbo system (BioRad, USA). Filters were blocked for at least 1 h at room temperature in Tween-20 TBS (TTBS) (TBS-Sigma, 0.1% Tween-20, pH 7.4) containing 5% nonfat dry milk. Reactive signals were revealed by ECL Star (EuroClone) or LiteAblot extended (EuroClone).
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