Protein levels were determined by western blotting as previously described (27 (link)). Briefly, 4.5 μg of extracted protein was electrophoresed in polyacrylamide gels and transferred to PVDF membranes. After blocking with PVDF-blocking reagent (TOYOBO, Osaka, Japan) for 1 h, the membranes were incubated with primary antibodies overnight, followed by incubation with secondary antibody for 1 h. Antibody information is presented in Supplementary Table 1 (see section on supplementary data given at the end of this article). The bands on the immunoblot were detected using the Amersham ECL Prime kit (GE Healthcare Japan), digitized using WSE-6100 LuminoGraph (ATTO, Tokyo, Japan), and quantified using CS Analyzer 4 software (ATTO).
Amersham ecl prime kit
Manufactured by GE Healthcare
Sourced in Japan
The Amersham ECL Prime kit is a chemiluminescent detection system used for the identification and quantification of target proteins in Western blot analysis. The kit contains reagents necessary for the detection of proteins labeled with horseradish peroxidase-conjugated antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf blocking reagent, by Toyobo (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
β actin, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx 4 20 precast gel, by Bio-Rad (1 mentions)
Lumi imager f1 workstation, by Roche (1 mentions)
T 25 tissue culture flasks, by Thermo Fisher Scientific (1 mentions)
Image lab 4, by Bio-Rad (1 mentions)
Criterion tgxtm precast gel, by Bio-Rad (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Tgf β2, by Abcam (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Uqcrc2, by Abcam (1 mentions)
Cox 4, by Abcam (1 mentions)
α tubulin, by Abcam (1 mentions)
Mtcoi, by Abcam (1 mentions)
Ndufb8, by Abcam (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Atp5a, by Abcam (1 mentions)
10 protocols using amersham ecl prime kit
1
Quantitative Protein Analysis by Western Blotting
2
Western Blot Analysis of Rat Germ Cells
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For Western blot analysis, we collected enriched mitotic (spermatogonia, SG), meiotic (spermatocytes, SC) and post-meiotic (spermatids, ST) germ cell populations and total testis (TT) samples from adult Sprague Dawley rats, and prepared protein extracts using standard protocols as published61 (link)–63 (link). 20 μg of total protein extract was loaded on a mini-PROTEAN TGX 4–20% precast gel (Bio-Rad) and transferred on PVDF membranes (Merck Millipore, ISEQ. 00010). The membranes are incubated with a rabbit polyclonal anti-EXOSC10 antibody (Abcam, ab50558; 1:800) overnight at 4 °C. After three washing steps, an HRP-conjugated goat anti-rabbit antibody was incubated for 1 h. Signals were revealed with an Amersham ECL Prime kit (GE Healthcare, RPN2232). An anti-β-tubulin antibody (Sigma Aldrich, T4026; 1:200) was used as a loading control.
3
Flavonoid Pre-Treatment Effects on Protein Expression
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MDA-MB231 cells and LECs were grown in T-25 tissue culture flasks (Nunc, Roskilde, Denmark) to 80% confluence and then pre-treated with the flavonoids for 0.5, 1, 2, and 4 h. Then, cells were processed for SDS gel electrophoresis and Western blotting as described before (Nguyen et al., 2015 (link)). Chemo-luminescence was developed by Amersham ECL prime Kit (GE Healthcare, Freiburg, Germany) and detected using a Lumi-Imager F1 Workstation (Roche, Basel, Switzerland). Densitometry of the Western blots was analyzed with the Image-J software (National Institutes of Health, Bethesda, MD, United States).
4
Quantification of PLN, SERCA2a in Heart
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Frozen hearts were homogenized in RIPA buffer (Thermo Fisher Scientific, Massachusetts, USA) containing plus proteinase inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland) and centrifuged at 20,000 x g for 10 min at 4°C. After a determination of protein concentrations, SDS-PAGE was performed using the supernatant with CriterionTM Precast Gels (10–20% Tris-Tricine/Peptide, BIO-RAD, California, USA) for PLN and CriterionTM TGXTM Precast Gels (4–20%, BIO-RAD) for SERCA2a and actin. They were transferred to PVDF membranes in the Trans-Blot Turbo (BIO-RAD) and blocked in PVDF Blocking Reagent (TOYOBO, Osaka, Japan). For immunoreaction, blots were incubated with 1: 1000 diluted monoclonal antibody to PLN (MilliporeSigma, Darmstadt, Germany), 1: 1000 diluted monoclonal antibody to SERCA2a (abcam, Cambridge, UK), or 1: 1000 diluted monoclonal antibody to actin (MilliporeSigma) at 4°C overnight. The antigens were detected by the luminescence method (Amersham ECL Prime kit, GE Healthcare, Little Chalfont, UK) with 1: 25000 diluted anti-mouse IgG (GE Healthcare) or 1: 25000 diluted anti-rabbit IgG (GE Healthcare) in the ChemiDoc (BIO-RAD). The intensity of bands was calculated by Image Lab 4.0 (BIO-RAD).
5
Western Blot Analysis of Cardiac Protein Expression
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Protein expression levels were determined by western blot analysis as previously described.26 (link)
Briefly, 10 μg of proteins extracted from cardiac tissues were electrophoresed in polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After 1-hour incubation with a blocking reagent, the membranes were incubated with primary antibodies overnight at 4°C, followed by secondary antibody for 1 hour at room temperature. The following antibodies were used for western blot analyses: TGF-β2 (Abcam Japan, Chuo, Tokyo, Japan; Product ID: ab36495, RRID: AB_778343; mouse monoclonal antibody; dilution: 1:2000; molecular weight [MW]: 25 kDa [bioactive dimer]), NHE-1 (Santa Cruz Biotechnology, Dallas, TX, USA; Product ID: sc-136239, RRID: AB_2191254; mouse monoclonal antibody; dilution: 1:500; MW: 110 kDa), β-actin (Santa Cruz Biotechnology; Product ID: sc-47778; RRID: AB_2714189; mouse monoclonal antibody; dilution: 1:10,000; MW: 43 kDa), and anti-mouse IgG from sheep (GE Healthcare Japan, Hino, Tokyo, Japan; Product ID: NA931; RRID: AB_ 772212; dilution: 1:20,000). The bands on immunoblots were detected using the Amersham ECL Prime Kit (GE Health Care Japan), digitized using a WSE-6100 LuminoGraph (ATTO, Taito, Tokyo, Japan), and quantified using CS Analyzer 4 software (ATTO).26 (link)
Briefly, 10 μg of proteins extracted from cardiac tissues were electrophoresed in polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After 1-hour incubation with a blocking reagent, the membranes were incubated with primary antibodies overnight at 4°C, followed by secondary antibody for 1 hour at room temperature. The following antibodies were used for western blot analyses: TGF-β2 (Abcam Japan, Chuo, Tokyo, Japan; Product ID: ab36495, RRID: AB_778343; mouse monoclonal antibody; dilution: 1:2000; molecular weight [MW]: 25 kDa [bioactive dimer]), NHE-1 (Santa Cruz Biotechnology, Dallas, TX, USA; Product ID: sc-136239, RRID: AB_2191254; mouse monoclonal antibody; dilution: 1:500; MW: 110 kDa), β-actin (Santa Cruz Biotechnology; Product ID: sc-47778; RRID: AB_2714189; mouse monoclonal antibody; dilution: 1:10,000; MW: 43 kDa), and anti-mouse IgG from sheep (GE Healthcare Japan, Hino, Tokyo, Japan; Product ID: NA931; RRID: AB_ 772212; dilution: 1:20,000). The bands on immunoblots were detected using the Amersham ECL Prime Kit (GE Health Care Japan), digitized using a WSE-6100 LuminoGraph (ATTO, Taito, Tokyo, Japan), and quantified using CS Analyzer 4 software (ATTO).26 (link)
6
Immunoblotting Analysis of OXPHOS and Mitochondrial Proteins
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Lymphoblast samples from P1 and two control lines (C1 and C2) were processed for immunoblotting as previously described [Webb et al., 2015 ]. The membrane was probed with mouse anti‐OXPHOS cocktail at 1:1,000 (MitoSciences, ab110411, Eugene, OR, USA) and with rabbit anti‐GAPDH at 1:5,000 (Sigma, G9545, St. Louis, MO, USA) used as a loading control. Antibodies were visualized using IRDye 800CW or IRDye 680RD secondary antibodies and the Odyssey Infrared Imaging System (LI‐COR Biosciences, Lincoln, NE, USA).
Total protein was extracted from fibroblasts and muscle tissue of P5 and fibroblasts of P4, separated on 12% SDS‐PAGE gels, transferred to PVDF membranes and analyzed by immunoblotting using primary antibodies to VARS2 (either custom‐made or from MitoSciences, Eugene, OR, USA), NDUFB8, MTCOI, MTCOII, COXIV, SDHA, UQCRC2, ATP5A, and VDAC (all from Abcam, Cambridge, UK). β‐actin (Sigma, G9545, St. Louis, MO, USA), GAPDH (Millipore, Burlington, MA, USA), and α‐tubulin (Abcam) were used as loading controls. Chemiluminescent signals were detected using the Amersham ECL Prime Kit (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and the ChemiDocTM MP Imaging System (Bio‐Rad, Hercules, CA, USA).
Total protein was extracted from fibroblasts and muscle tissue of P5 and fibroblasts of P4, separated on 12% SDS‐PAGE gels, transferred to PVDF membranes and analyzed by immunoblotting using primary antibodies to VARS2 (either custom‐made or from MitoSciences, Eugene, OR, USA), NDUFB8, MTCOI, MTCOII, COXIV, SDHA, UQCRC2, ATP5A, and VDAC (all from Abcam, Cambridge, UK). β‐actin (Sigma, G9545, St. Louis, MO, USA), GAPDH (Millipore, Burlington, MA, USA), and α‐tubulin (Abcam) were used as loading controls. Chemiluminescent signals were detected using the Amersham ECL Prime Kit (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and the ChemiDocTM MP Imaging System (Bio‐Rad, Hercules, CA, USA).
7
BV-2 Cell Infection and Protein Analysis
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Unless otherwise stated, BV-2 cells were infected with MNV at an m.o.i. of 5 TCID50 per cell, and 12 h post-infection the cells were harvested and lysed in RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % SDS]. Cell lysates were resolved by SDS-PAGE and then probed using anti-GAPDH (AM4300; Ambion), anti-NS7, anti-ATP5α (ab14748; Abcam), or a mouse monoclonal anti-VF1 (4K5; Abmart) antibodies. The anti-NS7 antibody was described previously [24 (link)]. Goat anti-rabbit (sc-2030; Santa Cruz) and goat anti-mouse (sc-2031; Santa Cruz) secondary antibodies conjugated with HRP were used for detection by chemiluminescence using the Amersham ECL Prime Kit (GE Healthcare). Biochemical fractionation of infected cells was performed as previously described [19 (link)].
8
Western Blot Analysis of Protein Ectodomain
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The purified ectodomains were analyzed by SDS-PAGE on a 15 % acrylamide gel and stained overnight with InstantBlue (ISB1L, Sigma-Aldrich). For Western blotting, proteins were transferred on a polyvinylidene fluoride membrane (IPVH20200, Millipore) for 2 h at 250 mA and the membrane was saturated with phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 11.9 mM phosphate, Biosolve) containing 5 % (w/v) fat milk and 0.05 % (v/v) Tween-20 for 90 min at 37 °C. The membrane was incubated overnight at 4 °C with a monoclonal anti-FLAG antibody (F3165, Sigma-Aldrich, 76 ng/ml in blocking buffer), washed 4 times with the blocking solution and incubated with a secondary antibody conjugated to horseradish peroxidase (40 ng/ml, 172–1011, Bio-Rad) for 1 h at room temperature. Finally, the membrane was washed twice in PBS containing 0.05 % (v/v) Tween-20, and twice in PBS alone. The immunoreactive bands were detected by chemiluminescence with the Amersham ECL Prime kit (GE Healthcare), and the signal recorded with a ChemiDoc XRS + system (Biorad) after 5 to 60 s of exposure.
9
Mitochondrial Protein Analysis by Western Blot
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Total cellular protein was extracted from patient and control fibroblasts (as well as transfected cell lines), size separated on a 10% separating gel by SDS-PAGE and transferred to a methanol-activated PVDF membrane. Immunoblotting was performed using primary antibodies to NDUFA9, NDUFB8, NDUFA13, SDHA, UQCRC2, MTCOI, MTCOII, COXIV and ATPB (all from Abcam), TRIT1 (GeneTex GTX120508) and β-actin (Sigma A5316) as a loading control and TOMM20 (Abcam) as a non-respiratory chain protein mitochondrial control. Chemiluminescent detection of the bands was achieved using the Amersham ECL Prime Kit (GE Healthcare) for signal development, following manufacturer's instructions and the membrane was viewed using the ChemiDocTMMP Imaging System (Bio-Rad).
10
Western Blot Analysis of Proteins
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Protein samples were heated at 95°C and separated by SDS-PAGE. Proteins were then transferred to PVDF membrane (Millipore) in Tris/Glycine buffer with 20% methanol in cold room. PVDF membrane was blocked in TBST (pH 7.5), containing 0.05% Tween-20 and 5% skim milk powder or bovine serum albumin. The membrane was then incubated with primary antibodies, including anti-PABPC1 (Abcam ab21060, Cell signaling 4992 or Santa Cruz sc32318 1:1000), anti-PABPC4 (Abcam ab76763), anti-c-Myc (Santa Cruz sc 40), anti-tubulin (Sigma-Aldrich T9028 1:5000) and anti-GFP (Clontech 632381 1:2000).
The membrane was then washed three times in TBST and incubated with goat-anti-rabbit (Jackson ImmunoResearch 111-035-046 1:5000) or goat-anti-mouse (Jackson ImmunoResearch 115-035-071 1:5000) for 0.5 h, washed again, developed with Amersham ECL prime kit (GE healthcare RPN2236), and imaged on an Alpha Innotech imaging system.
The membrane was then washed three times in TBST and incubated with goat-anti-rabbit (Jackson ImmunoResearch 111-035-046 1:5000) or goat-anti-mouse (Jackson ImmunoResearch 115-035-071 1:5000) for 0.5 h, washed again, developed with Amersham ECL prime kit (GE healthcare RPN2236), and imaged on an Alpha Innotech imaging system.
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