Cells were lysed in SDS lysis buffer (1% SDS, 50 mM Tris pH7.4, 150 mM NaCl, 10% glycerol and 5µl/ml benzonase nuclease) for 10 min on ice, heated at 90°C for 5 min and centrifuged at 14,000 rpm for 10 min. Proteins were separated by SDS-PAGE, transferred to methanol activated Immobilon-P 0.45 µm PVDF membrane, probed with appropriate primary and secondary antibodies, and developed using SuperSignal West Pico or Dura Chemiluminescent Substrates (Thermo Scientific).
Dura chemiluminescent substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Dura Chemiluminescent Substrate is a laboratory reagent designed for the detection and quantification of proteins in Western blot analysis. It provides a consistent and stable chemiluminescent signal, enabling the visualization and analysis of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico, by Thermo Fisher Scientific (12 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nupage novex bis tris gel, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protein a bead, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Takara Bio (1 mentions)
Anti myc, by Fortrea (1 mentions)
Anti gfp, by Abcam (1 mentions)
Mammalian cell lysis kit, by Merck Group (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Goat anti snail1, by Abcam (1 mentions)
Rabbit anti twist1, by Santa Cruz Biotechnology (1 mentions)
Nupage novex, by Thermo Fisher Scientific (1 mentions)
Image station 440cf, by Kodak (1 mentions)
Anti mcm7 141, by Santa Cruz Biotechnology (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Image studio 5, by LI COR (1 mentions)
Mops sds buffer, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Thermo Fisher Scientific (1 mentions)
Tubulin, by Merck Group (1 mentions)
12 protocols using dura chemiluminescent substrate
1
Cell Lysis and Protein Extraction Protocol
2
Detecting Protein-Protein Interactions in N. benthamiana
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The cDNAs of PARP1, PARP2, PARG1 and PARG2 were cloned into the GFP-tagged pGWB405 and/or myc-tagged pGWB417 Gateway destination vectors [81 (link)] and the resulting constructs were transformed into A. tumefaciens GV3101(pMP90). Leaves of 4–5 week-old N. benthamiana plants were agroinfiltrated with OD600 0.4 of the resulting A. tumefaciens strains, and some samples were then infiltrated two days later with 2 μg/ml of bleomycin solution. Tissues were harvested three days after agroinfiltration and total proteins were prepared in extraction buffer (50 mM Tris-HCl (pH7.5), 150 mM NaCl, 5 mM EDTA, 0.2% Triton X-100, 10% glycerol, and Sigma-Aldrich plant protease inhibitor cocktail at 1:100). Immunoprecipitation was carried out with anti-GFP (Abcam) at 4°C overnight followed by incubation with protein A beads (Thermo Scientific) for 1–2 h. The beads were washed three times with extraction buffer without protease inhibitors. The precipitated proteins were eluded with the SDS loading buffer, subjected to SDS-PAGE and immunoblotted with anti-myc (Covance) and anti-GFP (Clontech) antibodies, and detected using Supersignal West Pico or Dura chemiluminescent substrates (Thermo Scientific).
3
SDS-PAGE Western Blot Protocol
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Cells were lysed in an SDS lysis buffer (1% SDS, 50 mM Tris [pH 7.4], 150 mM NaCl, 10% glycerol, and 5 μL/mL Benzonase nuclease) for 10 min before heating at 70°C for 10 min. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, probed with appropriate primary and secondary antibodies, and developed using SuperSignal West Pico or Dura Chemiluminescent Substrates (Thermo Scientific).
4
Cell Lysis and Immunoblotting Protocol
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Detergent cell lysates were prepared in digitonin buffer (1% digitonin, 50 mM Tris pH 7.4, 100 mM NaCl, 1 mM PMSF, protease infibitors), Triton X‐100 buffer (1% Triton X‐100, 100 mM NaCl, 50 mM HEPES pH 7.4, 1 mM PMSF, protease inhibitors) or RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 1% NP40, 0.5% deoxycholate, protease inhibitors). Cells were lysed for 30 min on ice, and lysates were subjected to centrifugation at 16,900 × g for 10 min. Sample buffer was added to supernatant, and samples were heated at 75°C for 10 min, except for immunoblots for TRC8 and gp78, which were heated at 50°C for 20 min, and for MARCH6, which were heated at 37°C for 30 min.
Proteins were resolved on SDS–PAGE and transferred to PVDF membrane (Immobilon‐P). Membranes were blocked in 5% (w/v) skimmed milk powder in PBS containing 0.2% Tween 20 for 1 h at room temperature. Proteins were separated by SDS–PAGE, transferred to methanol activated Immobilon®‐P 0.45 μm PVDF membrane, probed with appropriate primary and secondary antibodies and developed using SuperSignal™ West Pico or Dura Chemiluminescent Substrates (Thermo Scientific).
Proteins were resolved on SDS–PAGE and transferred to PVDF membrane (Immobilon‐P). Membranes were blocked in 5% (w/v) skimmed milk powder in PBS containing 0.2% Tween 20 for 1 h at room temperature. Proteins were separated by SDS–PAGE, transferred to methanol activated Immobilon®‐P 0.45 μm PVDF membrane, probed with appropriate primary and secondary antibodies and developed using SuperSignal™ West Pico or Dura Chemiluminescent Substrates (Thermo Scientific).
5
Western Blot Analysis of Astrocyte Proteins
6
Nuclear Protein Expression Analysis
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Cells were treated for 96 h with 2 mM VPA, and at the end of the treatment, equal concentrations of cell lysate from nuclear fractions were obtained as already described [38 (link)], were electrophoresed on a 4–12% NuPAGE Novex Bis-Tris gel (Life Technologies by Thermo Fisher Scientific, Waltham, MA, USA) with MES (NuPage Novex, Life Technologies by Thermo Fisher Scientific, Waltham, MA, USA). Western blots were performed using standard procedures [38 (link)]. Primary antibodies used were: goat anti-Snail1 (1:1000, AbCam, Cambridge, UK), and rabbit anti-Twist1 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA). As loading control protein, rabbit anti-Histone 3 (1:2000, Sigma-Aldrich) was used. Proper HRP-conjugated secondary antibodies were used (1:2000 anti-rabbit, Bio-Rad, Hercules, CA, USA, 1:5000 anti-goat Santa Cruz Biotechnology). Proteins were visualised using SuperSignal West Pico or Dura chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA) with a Kodak Image Station 440 CF (Eastman Kodak Co., New Haven, CT, USA). Bands were then quantified with ImageJ (https://imagej.nih.gov/ij/ ) and results were normalised versus controls. The experiments were performed at least in triplicates.
7
Western Blot Analysis of Cell Lysates
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Cells were lysed in lysis buffer on ice (100 mM Tris/HCl, 150 mM NaCl, 1% Triton X, 1 mM EDTA, 1 mM DTT, 10 ml lysis buffer + 1 tablet protease inhibitor), scraped with a cell scraper, and snap frozen in liquid nitrogen. A volume of 100 μl of this mix was sonicated in a Bioruptor sonicator device (Diagenode), before 100 μl of 2x SDS loading dye were added. About 10 mg of protein extract were resolved by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked in 5% dried milk in 1x PBS plus 0.2% Tween 20 and incubated with the diluted primary antibodies overnight at 4°C. Secondary anti-rabbit or anti-mouse IgG antibodies conjugated to horseradish peroxidase (HRP) were used as the secondary antibodies. The HRP was detected by incubating the membrane with the SuperSignal West Pico or Dura Chemiluminescent Substrate (Thermo Scientific) for 4 minutes, before the signals were acquired on a digital chemiluminescence imaging system. The antibodies used for western blot analysis were anti-MCM7 (141.2 Santa Cruz), anti-Tubulin (ab18251 abcam), and anti-pAKT (44-621G Thermo Scientific). Quantification of western blots was done by measuring the band intensities in Fiji.
8
Western Blot Protocol for Protein Quantification
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Whole cell extracts for western blot analysis were prepared using 1× SDS sample buffer without DTT or bromophenol blue (125 mM Tris-base (pH 6.8), 20% glycerol and 2.5% SDS). Proteins were quantified using BCA protein assay kit (Thermo Fisher) according to the manufacturer's instructions. Samples were resolved on NuPAGE 4–12% Bis–Tris gels in MOPS-SDS buffer (Thermo Fisher) at 30 μg/lane. The sources and dilutions of the antibodies used for western blot analyses are listed in Supplementary Table S2 . HRP-conjugated secondary antibodies (Epigentek) were used at 1:10,000. Immunoblots were developed using SuperSignal West Pico or Dura chemiluminescent substrate (ThermoFisher), and imaged using a Kodak Processor M35A. The imaged films were scanned and quantified using ImageStudio 5.2 software (Li-Cor).
9
Western Blot Analysis of Cdk5 Signaling
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Tissue homogenates were lysed in tissue protein extraction reagent (Thermo Fisher Scientific) containing protease (cOmplete Mini) and phosphatase (PhosSTOP) inhibitor cocktail tablets (both Roche, Indianapolis, IN). Total protein concentration was measured using a Bradford Protein Assay (Bio-Rad, Hercules, CA). Protein of 40 µg was separated on a 4% to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Thermo Fisher Scientific), transferred to a 0.45-µm nitrocellulose membrane, and immunoblotted with antibodies directed against p39 (Abcam, Cambridge, MA), p35 (C-19), Cdk5 (C-8) (both Santa Cruz Biotechnology, Santa Cruz, CA), and tubulin (Sigma-Aldrich) overnight. Membranes were washed with phosphate-buffered saline plus 5% nonfat dry milk with 0.05% Tween 20 (Sigma-Aldrich) and then incubated for 1 h with 1:2000 dilution of anti-mouse/anti-rabbit secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The membranes were developed with SuperSignal West Pico or Dura Chemiluminescent Substrate (Thermo Fisher Scientific).
10
Western Blot Analysis of Pan02 Cells
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Protein was extracted from transduced Pan02 cells with a protein lysis buffer consisting of 2% SDS, 100mM Tris HCl, 100mM NaCl, 1X protease inhibitor (Thermo Fisher Scientific). Protein concentration was determined by BCA assay (Thermo Fisher Scientific) according to manufacturer’s protocols and samples were diluted to 20 μg/mL with reducing sample buffer (Thermo Fisher Scientific) and loaded into pre-cast 4 to 12% Bis-Tris Mini Protein Gels (Thermo Fisher Scientific). Proteins were transferred to a PVDF membrane in 1X TGE + 20% methanol, and blocked for 60 minutes in 5% milk in TBS + 0.1% Tween-20 (TBST). All antibodies were diluted 1:1000 in 5% BSA or 5% milk and incubated overnight at 4°C (CST and Abcam). Wash steps were performed using TBST and images were obtained with iBright imaging (Thermo Fisher Scientific) using an HRP-conjugated secondary antibody (CST) and SuperSignal West Pico or Dura Chemiluminescent Substrate (Thermo Fisher Scientific).
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