Cells were harvested, lysed, and processed for western blot analysis as described previously using 150mM NETN lysis buffer (20 mM Tris [pH 8.0], 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, and 10 mg/mL aprotinin). For cell fractionation, we isolated cytoplasmic and soluble nuclear fractions with the NE-PER Kit (Thermo Scientific) according to the manufacturer’s protocol; to isolate the chromatin fraction, the insoluble pellet was resuspended in RIPA buffer and sonicated in a BioRuptor according to the manufacturer’s protocol (high power, 15 min, 30 s on and 30 s off at 4°C). Proteins were separated using SDSPAGE and electrotransferred to nitrocellulose membranes. Membranes were blocked in 5% milk PBS-Tween and incubated with primary antibody for 1 hr. Abs for western blot analysis included anti-PCNA (Abcam), anti-H2B (Cell Signaling Technology), anti-β-actin (Sigma), anti-FANCJ (E67), anti-HLTF (Abcam), anti-SMARCA1 (Abcam), and anti-HLTF (Santa Cruz Biotechnology). Membranes were washed, incubated with horseradish-peroxidase-linked secondary antibodies (Amersham), and detected by chemiluminescence (Amersham).
Anti h2b
Manufactured by Cell Signaling Technology
Anti-H2B is a laboratory reagent that recognizes the histone H2B protein. It is used in various research applications that involve the detection and analysis of H2B, such as Western blotting, immunoprecipitation, and immunofluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence, by Cytiva (2 mentions)
Horseradish peroxidase linked secondary antibody, by Cytiva (2 mentions)
Anti smarca1, by Abcam (2 mentions)
Anti hltf, by Santa Cruz Biotechnology (2 mentions)
Ne per kit, by Thermo Fisher Scientific (2 mentions)
Anti hltf, by Abcam (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti pcna, by Abcam (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Total histone extraction kit, by Epigentek (1 mentions)
Anti lamin a c, by Cell Signaling Technology (1 mentions)
Anti phospho acc ser79, by Cell Signaling Technology (1 mentions)
Anti acc, by Cell Signaling Technology (1 mentions)
Ponceau s solution, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti cox 4, by Abcam (1 mentions)
Anti acetyl lysine, by Cell Signaling Technology (1 mentions)
Anti succinyllysine, by PTM Biolabs (1 mentions)
Anti malonyl lysine, by PTM Biolabs (1 mentions)
6 protocols using anti h2b
1
Cell Fractionation and Western Blot Analysis
2
Histone Extraction and Protein Analysis
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Cells and tissues are lysed and homogenized with RIPA buffer (Thermo Fisher Scientific, Cat. #89900) containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Cat. # PI78447), 5 M trichostatin A (Cayman Chem, Cat. #89730), and 5 mM nicotinamide (Sigma-Aldrich, Cat. #72340). Histones were extracted from cells and tissues using a total histone extraction kit (EpiGentek, Cat. #OP-0006). Before SDS-PAGE, protein samples were heated at 70°C for 5 min. Antibodies used are listed as follows: anti-β-tubulin (Cell Signaling Technology, Cat. #2128), anti-SIRT5 (Cell Signaling Technology, Cat. #8782), anti-acetyl-lysine (Cell Signaling Technology, Cat. #9814), anti-malonyl-lysine (PTM Biolabs, Cat. #PTM-901), anti-succinyl-lysine (PTM Biolabs, Cat. # PTM-401), anti-glutaryl-lysine (PTM Biolabs, Cat. #PTM-1151), anti-ACC (Cell Signaling Technology, Cat. #3676), anti-phospho-ACC (Ser79) (Cell Signaling Technology, Cat. #3661), anti-H2B (Cell Signaling Technology, Cat. #12364), anti-KAT2A (Cell Signaling Technology, Cat. #3305), anti-HSP60 (Abcam, Cat. #ab59457), anti-Lamin A/C (Cell Signaling Technology, Cat. #2032), anti-COX IV (Abcam, Cat. #ab16056), and anti-GAPDH (Cell Signaling Technology, Cat. #5174). Ponceau S solution (Sigma-Aldrich, Cat. #P7170) was used for protein staining.
3
Protein Extraction and Western Blot Analysis
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Total protein was extracted with RIPA buffer containing phenylmethylsulfonyl fluoride (PMSF) and Halt Protease and Phosphatase Inhibitor Cocktail. Membrane protein was extracted by membrane and cytosol protein extraction kit (Beyotime Biotechnology, China). The concentration of proteins was tested using the bicinchoninic acid (BCA) protein assay. Protein samples (30 μg) were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Bio-Rad, Richmond, CA, USA). Membranes were blocked in 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h at room temperature. Then, membranes were incubated with primary antibody at 4 °C overnight. Anti-MTPα, anti-IRS1/P-IRS1, anti-Glut4, anti-β-actin, anti-GAPDH, anti-SIRT1, anti-Ace, anti-Ub and anti- Na+-ATPase α-1 were from Abcam. Anti-Akt/P-Akt, anti-VDAC1, anti-H2B and secondary antibody were from Cell Signaling Technology. Probed membranes were washed several times with TBST, and then incubated with horseradish peroxidase conjugated secondary antibodies at room temperature for 1 h. Bound antibody was detected with enhanced chemiluminescence (Millipore, Billerica, MA, USA). Protein expression was quantified using Image J software (NIH, USA). Total protein expression was normalized with respect to β-actin/GAPDH expression and membrane protein was normalized with respect to Na+-ATPase α-1 expression.
4
Subcellular Fractionation and Immunoblotting
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Cardiomyocytes were fractionated using a subcellular protein fractionation kit for cultured cells (Thermo Fisher, Waltham, MA; cat # 78840) per the manufacturer‐provided protocol. Lysate was separated on gradient (4% to 12%) XT gels (Bio‐Rad, Hercules, CA) and probed for anti‐GR (3660S, Cell Signaling), anti‐GAPDH (97166S, Cell Signaling), and anti‐H2b (12364S, Cell Signaling).
5
Immunoblotting Analysis of Cellular Fractionation
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Cells were harvested and lysed with lysis buffer: 150 mM NaCl, 10 mM EDTA, 10 mM Tris, pH 7.4, 1% X-100 Triton. Cellular fractionation was performed according manufacturer’s instruction (FractionPREP, BioVision). Cell lysates were subjected to SDS-PAGE, transferred onto a pure nitrocellulose membrane (BioRad) and blocked with 5% fat-free milk. Primary antibodies for immunoblotting included: anti-AIF1 (1:1000, Cell Signaling) and and anti-H2B (1:1000, Cell Signaling) as loading control. Membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (1:10,000, Santa Cruz Biotechnology, cat#: sc-2005) or anti-rabbit secondary antibody (1: 10,000, AnaSpec Inc., cat#: AS-28177) for 1 h and chemi-luminescence signals were detected by HRP substrate.
6
Cell Fractionation and Western Blot Analysis
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Cells were harvested, lysed, and processed for western blot analysis as described previously using 150mM NETN lysis buffer (20 mM Tris [pH 8.0], 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, and 10 mg/mL aprotinin). For cell fractionation, we isolated cytoplasmic and soluble nuclear fractions with the NE-PER Kit (Thermo Scientific) according to the manufacturer’s protocol; to isolate the chromatin fraction, the insoluble pellet was resuspended in RIPA buffer and sonicated in a BioRuptor according to the manufacturer’s protocol (high power, 15 min, 30 s on and 30 s off at 4°C). Proteins were separated using SDSPAGE and electrotransferred to nitrocellulose membranes. Membranes were blocked in 5% milk PBS-Tween and incubated with primary antibody for 1 hr. Abs for western blot analysis included anti-PCNA (Abcam), anti-H2B (Cell Signaling Technology), anti-β-actin (Sigma), anti-FANCJ (E67), anti-HLTF (Abcam), anti-SMARCA1 (Abcam), and anti-HLTF (Santa Cruz Biotechnology). Membranes were washed, incubated with horseradish-peroxidase-linked secondary antibodies (Amersham), and detected by chemiluminescence (Amersham).
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