To isolate proteins, samples were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS (Sigma Aldrich, L3771), 1% NP-40 (Sigma Aldrich, NP-40S), 0.25% sodium deoxycholate (Sigma Aldrich, D6750), 1 mM EDTA, 1 mM sodium fluoride, 1 mM Na3VO4) with a protease inhibitor cocktail (Roche Life Science, 11 697 498 001). For immunoblot analysis, proteins were electrophoresed on SDS-polyacrylamide gels and transferred onto PVDF membranes. The membranes were blocked with 4% skim milk (BD Difco, 232100) in Tris-buffered saline (TBS; 50 mM Tris-Cl, 150 mM NaCl, pH 7.5) containing 0.5% Tween-20 (Sigma Aldrich, P1379; TBST) and subsequently incubated with anti-PRDX1 (Abcam, ab41906), anti-PRDX2 (AbFrontier, LF-PA0007), anti-PRDX3 (AbFrontier, LF-PA0030), anti-PRDX4 (AbFrontier, LF-PA0009), anti-NR1H3/LXRα (Abcam, ab41902), anti-LC3B (Cell Signaling Technology, 2775), anti-SQSTM1/p62 (Abcam, ab56416), anti-CD36 (Santa Cruz Biotechnology, sc-9154), anti-LMNB/laminB (Santa Cruz Biotechnology, sc-6217) and anti-GAPDH (Santa Cruz Biothechnology, sc-25778) primary antibody and horseradish peroxidase-conjugated secondary antibody (Millipore, AP106P, AP124P and AP124P). Immunoreactive bands were detected with ECL™ western blotting reagents (GE Healthcare Life Sciences, RPN2106).
Np40s
Manufactured by Merck Group
Sourced in United States
NP40S is a non-ionic detergent used in a variety of biological and biochemical applications. It is a mild detergent that is commonly used for cell lysis, protein extraction, and enzyme purification. The core function of NP40S is to solubilize and extract proteins from cells and tissues while maintaining the native structure and function of the proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lf pa0030, by AbFrontier (1 mentions)
Ecl western blotting reagent, by GE Healthcare (1 mentions)
L3771, by Merck Group (1 mentions)
Ap106p, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
Sodium fluoride, by Merck Group (1 mentions)
E1014, by Merck Group (1 mentions)
P8920, by Merck Group (1 mentions)
Duolink in situ detection kit, by Merck Group (1 mentions)
P9416, by Merck Group (1 mentions)
Fluorometric assay kit, by Abcam (1 mentions)
S8830, by Merck Group (1 mentions)
S6508, by Merck Group (1 mentions)
Ecl blotting substrate, by Advansta (1 mentions)
8 protocols using np40s
1
Protein Extraction and Immunoblot Analysis
2
Western Blot Analysis of PEL Cells
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1 × 106 PEL cells were washed twice with 1X PBS and centrifuged at 1500 rpm for 5 min. Cells were lysed in a 1X RIPA buffer containing 150 mM Nacl, 1% NP-40 (Sigma Aldrich, NP40S), 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid (Sigma Aldrich, D6750), 0.1% SDS (Sigma Aldrich, 71736), protease (Sigma Aldrich, St Louis, MO, USA; S8830) and phosphatase inhibitors (Sodium Orthovanadate; Sigma Aldrich, St Louis, MO, USA; S6508) (Sodium Fluoride; Sigma Aldrich, St Louis., MO, USA; S7920). Following this, 10 µg of protein lysates were subjected to electrophoresis on 10% or 15% acrylamide gels. Gels were transferred to nitrocellulose membranes (Bio-Rad, 162-0115) for 2 h in Tris-glycine buffer. Membranes were blocked in PBS-0.1% Tween 20 solution containing 3% BSA, probed with specific antibodies and developed using ECL Blotting Substrate (Advansta, K-12045-D20).
3
Nuclei Isolation and Chromatin Digestion Protocol
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48 or 36 hours after transfection, the cells were harvested (density 4–5 × 106 cells/ml), resuspended in 500 µl of lysis buffer (10 mM HEPES/KOH pH7.5, 1.5 mM MgCl2, 1 mM DTT, 10 mM EDTA, 10% glycerol and 1% Tergitol-type NP40 (Sigma NP40S) supplemented with proteinase inhibitors (Roche complete without EDTA)) and incubated for 10 minutes on ice. Then nuclei were pelleted by centrifugation at 5000xg for 5 minutes and the supernatant (mostly cytosol) was discarded. The pellet was resuspended in lysis buffer without EDTA but containing 1 M urea, incubated for 5 minutes on ice and again pelleted at 5000xg for 5 minutes. The urea washing step was carried out twice in total, then the nuclei were resuspended in 110 µl of lysis buffer without EDTA and without urea. To digest the chromatin, 250 U of benzonase (Merck Millipore E1014, 90% purity grade) were added and the resuspended nuclei were incubated at 37°C for 3 minutes in a heating block. The digestion was stopped by adding EDTA and NaCl to a concentration of 10 mM and 500 mM, respectively. The insoluble fraction was pelleted by centrifugation at 16000xg for 5 minutes and the supernatant was used as input material for the immunoprecipitation.
4
Corneal Stromal and Epithelial Chromophore Assay
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Corneas were photographed daily until day 6 via slit-lamp biomicroscopy before and after fluorescein instillation. Corneas were then excised, fixed in 4% PFA, permeabilized with 0.02% Tergitol (Sigma #NP40S) and 0.01% sodium deoxycholate (Sigma #30970), labeled for 16 h at room temperature in 0.1% X-Gal solution, DAPI-counterstained, and whole mounted for macroscopy examination. X-Gal-generated chromophore (X-Gal-c) release in the stroma was studied using 40-μm-interspaced Z-stack images taken at ×8 magnification. The epithelial and stromal chromophore coloration were identified by their positions in the Z stack. X-Gal-generated chromophore release was assessed for inoculated center and peripheral zones. Stromal X-Gal-generated chromophore release was quantified on maximum intensity Z-stack projections after epithelial signal subtraction. Yellow keratocytes were counted manually. Epithelial X-Gal-generated chromophore was quantified after subtracting stromal chromophore expression, and normalization to corneal area. Image analysis used ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/ ).
5
Proximity Ligation Assay for Protein Interactions
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Cells were seeded to a density of 50–70% on 12 mm glass coverslips coated in poly-L-lysine (Sigma-Aldrich, P8920). Cells were fixed with a 2% PFA solution (2% PFA, 0.2% Triton-X-100, pH 8.2), washed with 1X PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), permeabilized with 0.5% tergitol (Sigma-Aldrich, NP40S), and washed with 1X PBS. PLAs were performed according to manufacturer’s instructions using the Duolink In Situ Detection Kit (Sigma-Aldrich, DUO92013). Confocal microscopy was done at the SickKids Imaging Facility and foci counted using CellProfiler [106 (link)]. Signal intensity was obtained using ImageJ, with background signal subtracted for each figure [107 (link)].
6
CRISPR Genotyping by Sanger Sequencing
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For genotype analysis, cells were pelleted and resuspended in lysis buffer (0.45% NP-40 (NP40S, Sigma-Aldrich), 0.45% Tween-20 (P9416, Sigma-Aldrich), 0.2 mg mL−1 Proteinase K, 1× DreamTaq PCR buffer in water). Volume was adapted to cell amount, for one-quarter of a 96-well plate, 30–50 µL were used. For PCR, 1 µL of DNA containing lysate, 1× DreamTaq Green buffer, 0.2 mM dNTPs, 1 µM primer mix (Ex12F2 5′-CAGCATACTGCCTTGCAAATAA-3′, Ex12R2 5′-TGATTCCACAAAAATAATCCCAG-3′) and Taq polymerase were mixed and brought to 15 µL with H2O. Clones were screened for Tasor-null alleles using Sanger sequencing. Results were analyzed with TIDE (Brinkman et al. 2014 (link)).
7
Proteasome Activity Fluorometric Assay
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Proteasome activity was assessed using a fluorometric assay kit from Abcam (Waltham, MA, US, ab107921). Briefly, 2 × 106 cells were harvested by trypsinization, washed with cold PBS, and lysed in 60 µl 0.5% NP-40 (Sigma-Aldrich, St. Louis, MO, US, NP40S). Lysates were clarified by centrifugation at 13,000 rpm for 12mins at 4 °C then supernatants were separated and diluted 1:3 with the assay buffer. Samples were transferred into 96-well black assay plates and assayed per the manufacturer's instruction. The fluorescent signal was captured at different time intervals using a FlexStation3 Microplate Reader with excitation/emission wavelengths set to 350/440 nm.
8
Western Blot Analysis of PBMC Proteins
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A total of 5 × 106 PBMCs were washed twice with 1X PBS and centrifuged at 1500 rpm for 5 min. Cells were lysed in a 1X RIPA buffer containing 150 mM NaCl, 1% NP-40 (Sigma Aldrich; NP40S), 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid (Sigma Aldrich, D6750), 0.1% SDS (Sigma Aldrich, 71736), protease (Sigma Aldrich; S8830) and phosphatase inhibitors (Sodium Orthovanadate; Sigma Aldrich; S6508) (Sodium Fluoride; Sigma Aldrich; S7920). Then, 15 µg of protein lysates was subjected to electrophoresis on 4–20% SDS-PAGE gradient gels (NuSep, Germantown, MD, USA; NN12-420), according to the manufacturer’s instructions. Then, the gels were transferred to nitrocellulose membranes (Bio-Rad, 162-0115) for 2 h in Tris-glycine buffer. The membranes were blocked in PBS-0.1% Tween 20 solution containing 3% BSA, probed with specific antibodies, and developed using ECL Blotting Substrate (Advansta, San Josè, CA, USA; K-12045-D20).
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