Cells were washed two times with cold PBS and suspended in RIPA lysis buffer (Santa Cruz Biotechnology, Dallas, TX). After centrifugation, supernatant was collected, and the protein concentration was determined with the PierceTM BCA protein assay kit (Thermo Scientific). Ten or fifteen micrograms of protein from each sample was separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). To reduce nonspecific antibody binding, the membrane was blocked with Tris-buffered saline (TBS: 20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20) containing 5% bovine serum albumin, incubated with primary antibody at 4°C overnight, and washed with TBS. After incubation with peroxidase-labeled secondary antibody for 1 h at room temperature, the membrane was washed with TBS and analyzed with ImageQuant LAS4000 (GE Healthcare, Buckinghamshire, UK) using ECLTM Select Western Blotting Detection Reagent (GE Healthcare). Primary antibodies were used at 1:1000 dilution (anti-GDNF antibody), 1:2000 dilution (anti-Met antibody and anti-phospho-Met antibody) and 1:4000 dilution (anti-beta-actin antibody). Rat brain tissue was used as a positive control for GDNF expression.
Ecltm select western blotting detection reagent
Manufactured by GE Healthcare
Sourced in United States
The ECL Select Western Blotting Detection Reagent is a chemiluminescent reagent used for the detection of proteins in Western blot analysis. It provides a sensitive and reliable way to visualize and quantify target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Amersham protran 0.20 m nc nitrocellulose transfer membrane, by GE Healthcare (2 mentions)
Ecl na934 horseradish peroxidase donkey anti rabbit, by GE Healthcare (2 mentions)
Protran, by Cytiva (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (2 mentions)
Wet tank blotting system, by Bio-Rad (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Thermo Fisher Scientific (2 mentions)
Difco skim milk, by BD (2 mentions)
Xpert protease inhibitor cocktail solution, by GenDEPOT (2 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Amersham imager 680, by GE Healthcare (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Las 3000 imaging system, by Fujifilm (1 mentions)
Gapdh antibody, by Cell Signaling Technology (1 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
6 protocols using ecltm select western blotting detection reagent
1
Western Blot Analysis of GDNF and Met
2
Protein Production and Detection Protocol
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For protein production, 25 ml of PcMM or PdMM were inoculated with a final concentration of 106 conidia ml‐1 of transformants and were incubated at 25°C and 150 rpm for 5 (P. chrysogenum) or 11 days (P. digitatum). Total proteins from supernatants and purified PeAfpA and PdAfpB were separated by SDS‐PAGE (16% polyacrylamide gels) and transferred to Amersham Protran 0.20 µm NC nitrocellulose transfer membrane (GE Healthcare Life Sciences, Chicago, IL, USA) as described (Garrigues et al., 2018 (link)). Protein detection was accomplished using anti‐PeAfpA antibody diluted 1:2,500 (Garrigues et al., 2018 (link)) or anti‐PAFB antibody diluted 1:1,000 (Garrigues et al., 2017 (link)). As secondary antibody, 1 : 20 000 dilution of ECL NA934 horseradish peroxidase donkey anti‐rabbit (GE Healthcare Life Sciences) was used and chemiluminescent detection was performed with ECLTM Select Western blotting detection reagent (GE Healthcare Life Sciences) using Amersham Imager 680 (GE Healthcare Life Sciences). The experiments were repeated at least twice.
3
Quantitative Western Blot Analysis
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Cells were treated with cold sample lysis buffer (1% Triton X-100, Xpert Protease Inhibitor Cocktail Solution (GenDEPOT, Katy, TX, USA), 5 mM Ethylenediaminetetraacetic acid (EDTA, Thermo Fisher Scientific, Waltham, MA, USA), and 1 mM Phenylmethanesulfonyl fluoride (PMSF, Thermo Fisher Scientific, Waltham, MA, USA)). Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used for determining the protein concentration. An equal amount of protein was loaded for SDS-PAGE and transferred onto PVDF membrane using Wet/Tank Blotting Systems (Bio-Rad Laboratories Inc., Hercules, CA, USA). The membranes were incubated with 5% DifcoTM Skim Milk (BD Biosciences, San Jose, CA, USA) for 30 min at room temperature. The membranes were incubated with cleaved caspase-3 (Asp175) antibody (Cat. 9661S, Cell Signaling Technology, Danvers, MA, USA) and GAPDH antibody (Cat. sc-47724, Santacruz Biotechnology Inc., Dallas, TX, USA) overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). We added ECLTM Select Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) on the membrane for the detection of the signals from HRP. The images of protein bands were obtained by a LAS-3000 imaging system (Fujifilm, Minato, Tokyo, Japan).
4
Protein Extraction and Detection Protocol
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A total of 1 ml liquid culture or 100 mg mycelium from solid medium were harvested and mixed with 1 ml 1× ZAP (50 mM NaCl, 50 mM Tris–HCl pH 8.0, 10% glycerol, 10 mM PMSF). The subsequent disruption of the cells was done using 200 μl glass beads (0.4 mm diameter) and the FastPrep-24 instrument (MP Biomedicals) for 4 × 1 min at 5.5 m/s. Mycelial debris was removed by centrifugation at 4°C. Protein concentration of the supernatant was determined by Bradford assay (Bradford, 1976 (link)). Twenty to fifty μg of crude extract were separated by a 6% or 12% SDS-PAGE. 0,5% 2,2,2-trichloroethanol (TCE) in the polyacrylamide gels allowed fluorescent detection of proteins using the ChemiDocTM MP Imaging System (Bio-Rad). Gels were blotted using the Trans-Blot® TurboTM Transfer System (Bio-Rad). Membranes were blocked for 1 h in 2% ECLTM Blocking Agent (GE Healthcare) in TBS-T (20 mM Tris base, 150 mM NaCl and 0.1% Tween 20), followed by incubation with a Monoclonal ANTI-FLAG® M2 antibody (Sigma-Aldrich #F1804; 1:50000 in 2% ECLTM Blocking Agent in TBS-T) for 1 h at RT and 3 × 10 min wash in TBS-T. Blots were developed using ECLTM Select Western Blotting Detection Reagent (GE Healthcare) and signals detected with the ChemiDocTM MP Imaging System. Protein bands were quantified using Image Lab Software 6.0.1. Total protein was used as loading control.
5
Protein Extraction and Western Blotting
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Cells were treated with ice-cold sample lysis buffer consisting of 1% Triton X-100 (Sigma-Aldrich), 5 mM ethylenediaminetetraacetic acid (EDTA, Thermo Fischer Scientific), 1 mM phenylmethanesulfonylfluoride (PMSF, Thermo Fischer Scientific), and Xpert Protease Inhibitor Cocktail Solution (GenDEPOT, USA) in DPBS. Protein extracts were quantified with Protein Assay Dye Reagent Concentrate (Bio-Rad). An equal amount of total protein was separated by SDS-PAGE. All samples were then transferred to PVDF membrane (Bio-Rad) using a Wet/Tank Blotting System (Bio-Rad). The membranes were incubated first with blocking solution (Difco™ Skim milk, BD, USA), then primary antibodies were added, followed by the addition of horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, USA). For detection of the oxidized signals from HRP, we added substrates (ECLTM Select Western Blotting Detection Reagent, GE Healthcare, USA). The HRP images of protein bands were acquired by a LAS-3000 Imager (Fujifilm, Japan). Primary antibodies used in this experiment are listed in Supplementary Table S2 .
6
Immunodetection of Apple Antifreeze Proteins
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PdAfpB and the three PeAFPs were produced and puri®ed as previously described [22, 28] . Total proteins from supernatants and apple tissues, and puri®ed AFPs were separated by SDS-16 % polyacrylamide gels and transferred to Amersham Protran 0.20 µm NC nitrocellulose transfer membrane (GE Healthcare Life Sciences, Little Chalfont, United Kingdom). Protein detection was accomplished using anti-PeAfpA, anti-PeAfpB and anti-PeAfpC antibodies [22] diluted 1:2,500 for anti-PeAfpA and anti-PeAfpC and 1:1,500 for anti-PeAfpB. For PdAfpB detection anti-PAFB antibody diluted 1:1,000 was used [29] . As secondary antibody, 1:20,000 dilution of ECL NA934 horseradish peroxidase donkey anti-rabbit (GE Healthcare) was used and chemiluminescent detection was performed with ECL TM Select Western blotting detection reagent (GE Healthcare) using a LAS-1000 instrument (Fuji®lm, Tokyo, Japan). The experiments were repeated at least twice.
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