Whole-cell lysates for immunoblotting were prepared by dissolving cells in Laemmli Sample Buffer containing 5% 2-mercaptoethanol (Bio-Rad). Protein samples were loaded on 4–15% polyacrylamide gels (BioRad) and transferred onto 0.45 µm pore-size nitrocellulose membranes (Bio-Rad) using the Turboblot (BioRad). After that, blots were blocked with 5% milk for 1 h at room temperature. For phospho-tau, 5% BSA in TBS was used for blocking. Blots were incubated overnight at 4 °C with primary antibodies. And then blots were probed with secondary antibodies for 1 h at room temperature before ECL development and imaging (Bio-Rad). The primary antibodies used for immunoblotting are as follows: Lamin A/C antibody, (Abcam, 1:750); Lamin B1 antibody (Santa Cruz, 1:200); APP antibody (BioLegend, 1:400), total tau antibody (Santa Cruz, 1:200), PHF6 p-tau antibody (Santa Cruz, 1:200), γ-H2AX antibody (Abcam, 1:3000), and β-actin (1:5000, Sigma-Aldrich).
0.45 m pore size nitrocellulose membrane
Manufactured by Bio-Rad
0.45 µm pore-size nitrocellulose membranes are a type of filtration material used in laboratory applications. These membranes have a pore size of 0.45 micrometers, which allows for the effective separation and retention of particles, cells, and macromolecules during various filtration processes.
Automatically generated - may contain errors
Lab products found in correlation
Turboblot, by Bio-Rad (2 mentions)
β actin, by Merck Group (2 mentions)
Ecl development and imaging, by Bio-Rad (2 mentions)
Polyacrylamide gel, by Bio-Rad (1 mentions)
2 mercaptoethanol, by Bio-Rad (1 mentions)
γh2ax antibody, by Abcam (1 mentions)
Lamin b1 antibody, by Santa Cruz Biotechnology (1 mentions)
Lamin a c antibody, by Abcam (1 mentions)
S6 ribosomal protein, by Cell Signaling Technology (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Lamin a c, by Merck Group (1 mentions)
Total β catenin, by Cell Signaling Technology (1 mentions)
Non phospho active β catenin, by Cell Signaling Technology (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Luminata forte, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
4 protocols using 0.45 m pore size nitrocellulose membrane
1
Immunoblotting of Cell Lysates
2
Whole-Cell Lysates and Fractionation for Immunoblotting
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Whole-cell lysates for immunoblotting were prepared by dissolving cells in Laemmli Sample Buffer containing 5% 2-mercaptoethanol (Bio-Rad, Hercules, CA, USA). Nuclear and cytosolic fractions of iPSCs-differentiated cells were acquired using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA, USA #78833) as per the manufacturer’s instructions. Protein samples were loaded on 10–12% polyacrylamide gels and transferred onto 0.45 µm pore-size nitrocellulose membranes (Bio-Rad) using the Turboblot (BioRad). Blots were incubated overnight at 4 °C with primary antibodies and then probed with secondary antibodies before ECL development and imaging (Bio-Rad). The primary antibodies used for immunoblotting are as follows: lamin A/C (1:500, Millipore MAB3211), progerin (1:500, Cao et al., 2011), ∆Np63 (1:500, Biolegend, San Diego, CA, USA #619001), K14 (1:500, Invitrogen MA5-11599), K8 (1:500, Biolegend #904804), K18 (1:1000, Cell Signaling #4548), total β-Catenin (1:1000, Cell Signaling #9562), Non-phospho (Active) β-Catenin (1:1000, Cell Signaling #8814), RCC1 (1:1000, Cell Signaling #3589), S6 Ribosomal Protein (1:1000, Cell Signaling #2217), LEF1 (1:250, Santa Cruz sc-374412), and β-actin (1:1000, Sigma-Aldrich, St. Louis, MO, USA A3854).
3
Western Blot Analysis of Protein Lysates
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Total cell lysates from infection and transfection experiments were prepared in cold lysis buffer (1% Triton X-100, 1% NP-40 in PBS, pH 7.4) containing a cocktail of protease (Roche Applied Science, Mannheim, Germany) and phosphatase (Sigma-Aldrich) inhibitors. Protein concentration was determined by the DC protein assay (Bio-Rad Laboratories Inc., Hercules, CA, USA). Samples of 10–20 μg were diluted in 4x Laemmli buffer (Bio-Rad) with β-mercaptoethanol (Sigma-Aldrich), denaturated at 95 °C for 5 min, separated onto 10% SDS-PAGE gels, and transferred onto 0.45 µm pore-size nitrocellulose membrane (Bio-Rad). Membranes were blocked with 5% non-fat milk in Tween 0.1%–PBS or with 5% BSA in PBS, incubated with primary and horseradish peroxidase (HRP)-conjugated secondary antibodies, washed several times with TBS–0.5% Tween 20, and detected with a chemiluminescent HRP detection reagent (Luminata FORTE, Merck Millipore, Darmstadt, Germany or Clarity™ Western ECL Substrate, Bio-Rad). Bands were quantified by densitometric analysis using Quantity One® software (Bio-Rad).
4
Turbot Myeloperoxidase Protein Analysis
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Total protein was extracted from turbot white blood cells (WBC), serum and peritoneal fluid by using Cell Extraction Buffer (Life Technologies) and was then resolved in a 4-20%
Mini-PROTEAN® TGX™ gel (Bio-Rad) under non-reducing and reducing conditions.
Lyophilized myeloperoxidase (dissolved in distilled water) was also separated under reducing conditions. Proteins were transferred with a Trans-Blot SD semi-dry transfer cell (BioRad) onto a 0.45 µm pore size nitrocellulose membrane (Bio-Rad).
The membrane was blocked with blocking buffer and incubated with anti-turbot myeloperoxidase mouse serum antibodies (1:400), overnight at 4 o C. The membrane was then incubated with peroxidase-labelled rabbit anti-mouse Ig (1:1000) (Dako) for 1 h at room temperature, and, finally, with 0.06% 3,3'-diaminobenzidine tetrahydrochloride-nickel chloride (Sigma) and 0.003% H2O2 for 10 min. Negative samples included lanes without protein sample or without primary antibody. Lanes were recorded photographically with a Panasonic camera DMC-TZ4.
White blood cell collection, purification of myeloperoxidase and preparation of myeloperoxidase antiserum were carried out as previously described (by respectively Couso et al., 2001 and (link)Castro et al., 2008a) .
Mini-PROTEAN® TGX™ gel (Bio-Rad) under non-reducing and reducing conditions.
Lyophilized myeloperoxidase (dissolved in distilled water) was also separated under reducing conditions. Proteins were transferred with a Trans-Blot SD semi-dry transfer cell (BioRad) onto a 0.45 µm pore size nitrocellulose membrane (Bio-Rad).
The membrane was blocked with blocking buffer and incubated with anti-turbot myeloperoxidase mouse serum antibodies (1:400), overnight at 4 o C. The membrane was then incubated with peroxidase-labelled rabbit anti-mouse Ig (1:1000) (Dako) for 1 h at room temperature, and, finally, with 0.06% 3,3'-diaminobenzidine tetrahydrochloride-nickel chloride (Sigma) and 0.003% H2O2 for 10 min. Negative samples included lanes without protein sample or without primary antibody. Lanes were recorded photographically with a Panasonic camera DMC-TZ4.
White blood cell collection, purification of myeloperoxidase and preparation of myeloperoxidase antiserum were carried out as previously described (by respectively Couso et al., 2001 and (link)Castro et al., 2008a) .
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