Western blotting analysis was performed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Samples containing 30 μg of proteins were loaded into 12.5% SDS-PAGE gel. Then, samples were transferred to PVDF membranes using a semi dry transfer apparatus (Trans-blot Turbo, Bio-Rad Laboratories, Hercules, CA, USA). Membranes were treated with a blocking solution, containing 5% of non-fat dry milk in 1X TBS-T buffer (10 mM Tris-HCl, 100 mM NaCl, 0.1% Tween, pH 7.5), for 1 h and then incubated overnight with primary antibodies at 4 °C. Immunoreactivity was detected using the donkey anti-rabbit or anti-mouse secondary peroxidase-conjugated (GE Healthcare, Chicago, IL, USA). The immunoreactive bands were visualized using the enhanced chemiluminescence detection kit (ECL Advance, GE Healthcare, Chicago, IL, USA). The following antibodies were used for Western blot analysis: mouse monoclonal anti-mtTFA (sc-376672, Santa Cruz Biotechnology, Dallas, TX, USA, dilution 1:500), mouse monoclonal anti-β-Actin (66009-1-Ig, Proteintech, Rosemont, IL, USA, dilution 1:10,000), rabbit monoclonal anti-TOM20 (#42406, Cell Signaling Technology, Danvers, MA, USA, Rosemont, IL, USA, dilution 1:1000), Total OXPHOS Rodent WB Antibody Cocktail (ab110413, Abcam, Cambridge, UK, dilution 1:500) and rabbit polyclonal anti-GAPDH (GTX100118, GeneTex, Irvine, CA, USA, dilution 1:10,000).
Ecl advance
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
ECL Advance is a chemiluminescent detection system designed for the analysis of protein and nucleic acid targets in Western blotting and other blotting applications. It provides sensitive and reproducible detection of target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Anti rabbit or anti mouse secondary antibody, by Santa Cruz Biotechnology (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Luminol, by Santa Cruz Biotechnology (3 mentions)
Polyvinylidene difluoride membrane, by Cytiva (3 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (3 mentions)
Quantity one, by Bio-Rad (3 mentions)
Ecl advance blocking agent, by GE Healthcare (3 mentions)
Trans blot turbo, by Bio-Rad (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Polyacrylamide gel, by Lonza (2 mentions)
α tubulin, by Merck Group (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Sds lysis buffer, by Cell Signaling Technology (2 mentions)
Supersignal west pico, by Thermo Fisher Scientific (2 mentions)
Las 3000, by Fujifilm (2 mentions)
Anti erk, by Cell Signaling Technology (2 mentions)
Rabbit anti p erk, by Cell Signaling Technology (2 mentions)
0.2 μm nitrocellulose membrane, by Bio-Rad (2 mentions)
Las 3000 imager, by Fujifilm (2 mentions)
52 protocols using ecl advance
1
Western Blot Analysis of Mitochondrial Proteins
2
Western Blot Analysis of Protein Expression
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Protein was extracted from cells or aortic tissues using a protein extraction kit containing the protease inhibitor phenylmethanesulfonyl fluoride and protein phosphatase inhibitor. The concentration was determined using a BCA Protein Assay Kit (Beyotime, P0010). Proteins were denatured at 100°C for 10 min, electrophoretically separated on a 10% SDS-PAGE gel, and transferred onto a nitrocellulose membrane. Membranes were blocked with 10% bovine serum at room temperature for 1 h. Membranes were then incubated with the primary antibodies at 4°C overnight. Membranes were washed three times for 15 min and probed with a secondary antibody for 1 h at room temperature. Then, the membranes were washed three times for 15 min again. The bands were detected using enhanced chemiluminescence (ECL Advance; no. RPN2235, GE Healthcare Life Sciences). Finally, the signals were recorded using a ChemiDoc imaging system (Bio-Rad Laboratories). Tubulin or β-actin was used as a negative control. Details about the primary antibodies are available in Table S5 .
3
Western Blot Analysis of Signaling Proteins
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30 μg of protein were separated on polyacrylamide gels (Lonza), transferred to nitrocellulose membranes (Invitrogen) and blocked for 1 hour in blocking solution (2.5% milk powder (Biorad) in PBS containing 0.1% Tween-20 (PBS-T)). Membranes were incubated at 4°C overnight with primary antibody at a 1:1000 dilution in blocking solution (unless otherwise stated) against p-HER2tyr1221/1222, p-EGFRtyr1173, AKT, p-AKTser473, ERK, pERKthr202/tyr204, eEF2, p-eEF2thr56, mTOR, p-mTORser2448, eEF2k, p-eEF2kser366 (Cell Signaling Technology), p-eEF2kser359 (1:200) (SantaCruzBiotechnology), HER2 (Calbiochem), EGFR (1:250) (Neomarkers), and α-tubulin, anti-mouse and anti-rabbit secondary antibodies (Sigma-Aldrich). Detection was performed using Luminol (SantaCruzBiotechnology) or ECL Advance (GE Healthcare).
4
Western Blot Analysis of Signaling Pathways
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Cells were grown to log-phase, washed twice with ice-cold PBS, and lysed in SDS lysis buffer according to the manufacturer's protocol (Cell Signaling Technology, Danvers, MA, USA). Protein concentration was quantified using the BCA protein assay reagent bicinchoninic acid (Sigma). Cell lysates containing 25 μg of protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were immunoblotted with antibodies against phospho-Akt (S473), total Akt, phospho-p70S6K (T389), total p70S6K, phospho-rpS6 (S235/236), total rpS6, phospho-ERK (T202/Y204), total ERK, (all from Cell Signaling Technology), tubulin (Sigma), CK5 (Leica), and actin (Millipore), using SuperSignal West Pico (Thermo Scientific, Waltham, MA, USA) or ECL advance (GE Healthcare, Auckland, New Zealand) chemiluminescence reagents. Antibody reactivity was visualized using the chemiluminescence detection system by Fujifilm Las-3000.
5
Western Blot Analysis of Gal3 Protein
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Protein sample preparation and western blot analysis were carried out as described previously.8 (link) Briefly, whole cell extracts were lysed in SDS lysis buffer and were resolved on SDS-PAGE gels, and then transferred to a nitrocellulose membrane. Proteins were detected using rat monoclonal antibody against Gal3 (1:500, ascites fluid of TIB-166, ATCC# M3/38.1.2.8 HL.2) and beta-actin (1:5000 AC-15, Sigma,A5316). Protein bands were visualized using an enhanced chemiluminescence detection kit (ECL Advance, GE Healthcare, Marlborough, MA, USA).
6
Western Blot Protein Analysis Protocol
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Cells were lysed in NETN buffer (0.5% NP-40, 1 mM EDTA, 50 mM Tris-Cl (pH 8.0), NaCl (120 mM) containing protease (Calbiochem, San Diego, CA, USA) and phosphatase inhibitors (Sigma-Aldrich). Proteins were separated by SDS gel electrophoresis and transferred to 0.2 μM nitrocellulose membranes (Bio-Rad Laboratories, Hertfordshire, UK) overnight at 4 °C. Membranes were blocked for 6 h (0.15M NaCl, 1% milk and 0.1% Tween 20) and incubated with primary antibodies (diluted in blocking buffer) overnight. Following three washes (88 mM Tris, pH 7.8, 0.25% dried milk, and 0.1% Tween 20), membranes were incubated with a species-specific horseradish peroxidase–conjugated secondary antibody (in wash buffer) for 1 h at room temperature and washed a further three times, each for 10 min. Immunoreactive proteins were visualised using enhanced chemiluminescence (ECL Advance, GE Healthcare). All primary immunoblot images are available in Fig. S5 .
7
Protein Expression Analysis of Cultured Keratinocytes
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Routine procedures were followed (10 (link), 25 (link), 26 (link), 35 (link)). Briefly, cultured keratinocytes and dissected dorsal skin (0.5 × 0.5 cm, the area that received the treatment) were protein-extracted in radioimmunoprecipitation assay (RIPA, Sigma) buffer and electroblotted to nitrocellulose membranes after gel separation of proteins in a 4–15% polyacrylamide gel (Bio-Rad). Membranes were blocked with 5% BSA (Sigma) in TBST, and pERK and ERK were specifically detected with primary antibodies (rabbit anti-pERK (catalog #9101) and anti-ERK (catalog #4695), both at 1:2000; Cell Signaling Technology), secondary antibody (anti-rabbit peroxidase-conjugated, 1:5000; Jackson ImmunoResearch), and chemiluminescence substrate (ECL-Advance, GE Healthcare). Abundance was quantified using ImagePro Plus software. β-Actin, as a control, was detected with a mouse monoclonal anti-β-actin antibody (1:4000; catalog #sc-47778, Santa Cruz) or a rabbit polyclonal anti-β-actin antibody (1:4000; catalog #A5316, Sigma). Immunoblot band intensity was quantitated using the software Image J (National Institutes of Health).
8
Western Blotting of 3T3-L1 Cell Lysates
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3T3-L1 cells were harvested and lysed in ice-cold lysis buffer containing a protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride for 30 min, followed by centrifugation at 10,000 × g for 30 min at 4 °C. Proteins (50 μg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Amersham, UK). The membranes were incubated with primary antibodies, followed by incubation with anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology) and protein bands were visualized using an enhanced chemiluminescence system (ECL Advance, GE Healthcare, Hatfield, UK).
9
Western Blot Analysis of Protein Expression
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HeLa cells and murine liver tissue (in FastPrep-24 lysing matrix tubes; MP Biomedicals, Santa Ana, CA, USA) were lysed using radio-immunoprecipitation assay buffer (50 mM Tris–HCl, pH 7.4, 1% NP40, 0.25% Na-deoxycholate, 150 mM NaCl, and 1 mM EDTA) containing protease (Calbiochem, San Diego, CA, USA) and phosphatase inhibitors (Sigma–Aldrich Corp.). The proteins were separated by SDS gel electrophoresis and transferred to 0.2 μM nitrocellulose membranes (Bio-Rad Laboratories) overnight at 4 °C. The membranes were blocked for 6 h (0.15 M NaCl, 1% milk, and 0.1% Tween 20) and incubated with primary antibodies (diluted in blocking buffer) overnight. Following three washes (88 mM Tris, pH 7.8, 0.25% dried milk, and 0.1% Tween 20), membranes were incubated with a species-specific HRP-conjugated secondary antibody (in wash buffer) for 1 h at room temperature and washed a further three times, each for 10 min. Immunoreactive proteins were visualised using ECL Advance (GE Healthcare).
10
Glycoprotein CLEC4M Expression Analysis
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Cells and human liver tissues were lysed using urea buffer (7 M urea, 2 M thiourea, 3 % CHAPS, 3 % Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 30 mM Tris-HCl, pH 7.4). Protein (50 μg) was treated with N-glycanase (PNGase F) included in the Enzymatic Protein Deglycosylation Kit (Sigma-Aldrich, Saint Louis, MO). Treated and untreated proteins (20-μg samples) were electrophoresed in an SDS-10 % polyacrylamide gel and then transferred to a nitrocellulose membrane. CLEC4M protein was detected using 0.5 μg of goat anti-CLEC4M antibody N17 (Santa Cruz Biotechnology. Inc., Santa Cruz, CA) and 7.5 ng of horseradish-peroxidase-conjugated anti-goat IgG (Zymed Laboratories, San Francisco, CA) per ml. β-actin protein, used as an internal loading standard, was detected using 1 μg of mouse monoclonal anti-β-actin antibody (Abcam, Cambridge, UK) and 0.1 μg of horseradish-peroxidase-conjugated anti-mouse IgG (IBL, Gunma, Japan) per ml. CLEC4M and β-actin were detected using ECL Advance and Plus Western Blotting Detection Systems (GE Healthcare, Buckinghamshire, UK), respectively. Chemiluminescence was detected with a Light-Capture AE-6972 (ATTO, Tokyo, Japan) and analyzed using CS Analyzer version 2.07 software (ATTO).
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