Total protein from cell supernatant, chondrocyte and cartilage was prepared by RIPA buffer, the nuclear protein was isolated by using Nuclear Protein Extraction Kit (Beyotime) strictly in accordance with the Kit instructions, the protein mixtures were separated by gel electrophoresis in sodium dodecyl sulfate-polyacrylamide, which were then translated to nitrocellulose membranes. Briefly, 10% SDS-PAGE was used to extract 15μg plasma proteins from the cartilage tissue and chondrocytes. In addition to that, the proteins were electroblotted onto a polyvinylidene difluoride membrane with a thickness of 0.45 mm (Millipore, Bedford, MA, USA), which were then blocked via 5% non-fat dry milk in Tris-buffered saline with Tween 20 for one hour. Incubation of these membranes was then performed at 4 °C overnight with Klotho, Phos-GSK3β (S9), GSK3β, MMP2, MMP9, Wnt1, Wnt4, Wnt7 and β-catenin antibodies at room temperatures for one hour. To visualize antigen-antibody complexes, the enhanced chemiluminescence assay (Thermo Scientific, Pierce, Rockford, IL, USA) was applied.
Nuclear protein extraction kit
Manufactured by Beyotime
Sourced in China
The Nuclear Protein Extraction Kit is a laboratory tool designed to isolate and extract nuclear proteins from biological samples. It provides a standardized and efficient method for the separation of nuclear proteins from other cellular components. The kit includes reagents and protocols to facilitate the extraction process.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (12 mentions)
Ripa lysis buffer, by Beyotime (9 mentions)
Bca protein assay kit, by Beyotime (7 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Protease inhibitor, by Beyotime (4 mentions)
Vimentin, by Cell Signaling Technology (3 mentions)
Lysis buffer, by Beyotime (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Santa Cruz Biotechnology (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Lamin b1, by Abcam (3 mentions)
Odyssey infrared imaging system, by LI COR (3 mentions)
Cytoplasmic protein extraction kit, by Beyotime (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Phosphatase inhibitor, by Beyotime (3 mentions)
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Histone h3, by Cell Signaling Technology (2 mentions)
Gapdh, by Proteintech (2 mentions)
100 protocols using nuclear protein extraction kit
1
Protein Profiling of Chondrocytes and Cartilage
2
Transcription Factor Binding Assay
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Nuclear extracts were prepared by using Nuclear Protein Extraction Kit (Beyotime, Shanghai, China). Two sets of double-strand oligonucleotides with or without biotin-label centered on the rs71630754 were synthesized by TaKaRa Bio (Kyoto, Japan). EMSA/Gel-Shift Kit (Beyotime) was used to detect the DNA binding ability. Nuclear extracts were incubated for 20 min with unlabeled probes at a gradient concentration. For the super-shift assay, we first used JASPAR (https://jaspar.genereg.net/ ) to predict the possible binding transcription factors of the rs71630754. And gradient concentration of transcription factor 3 (TCF3) antibody (ab228699, Abcam, Cambridge, MA, USA) was incubated together with the nuclear extract and labeled probes for 20 min. After that we added the labeled oligonucleotide probes at room temperature, those reaction mixtures were loaded on an 8% polyacrylamide electrophoresis gel. The result was detected by SuperSignal West Femto Trial Kit (Thermo, Rockford, IL, USA).
3
Analyzing Apoptosis Signaling Pathways
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The lower lobe of the left lung was homogenized in lysis buffer, and the proteins were collected. For the detection of Nrf2 and NF-κB, the cytoplasmic component and nuclear component were isolated by treating a nuclear protein extraction kit (Beyotime Biotechnology, Shanghai, China) and centrifuged at 12,000 g for 10 min at 4°C. After the protein concentration was measured by a BCA kit, 50-μg protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked in 5% non-fat dry milk, followed by incubation with pro-caspase-3 (1:8,000, Abcam, United States), cleaved-caspase-3 (1:500, Abcam, United States), pro-caspase-9 (1:800, Abcam, United States), cleaved-caspase-9 (1:1,000, Abcam, United States), Nrf2 (1:5,000, Abcam, United States), and NF-κB (1:3,000, Abcam, United States) primary antibodies overnight at 4°C. After being washed and incubated with secondary antibody (1:5,000, Zhongshan Golden Bridge Biotechnology, Beijing, China) at room temperature for 1 h, the proteins were visualized with the enhanced chemiluminescence reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA, United States), analyzed using ImageJ version 1.61 software (National Institutes of Health, Bethesda, MD, United States) and normalized to β-actin and Lamin B.
4
Western Blot Analysis of Radiation-Treated Cells
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TSCC cells were seeded in six-well plates at a density of 1 × 105 cells/ml for attaching overnight. Then, cells were treated with IR or PL alone or PL (pretreated for 1 hr) + IR. After 24 hr, cells were washed with PBS and lysed with RIPA buffer. Nuclear proteins were extracted using the nuclear protein extraction kit (Beyotime Co., China). Protein concentrations were measured by using the BCA protein assay kit (Beyotime Co., China). Equal amounts of protein samples were electrophoresed on 8–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto an immobilon PVDF membrane at 100 V for 2 hr at 4°C. Membranes were probed with primary antibodies overnight at 4°C and then blotted with secondary antibodies for 2 hr. Super ECL Star (US Everbright, Inc.) and the UVP ChemStudio Imaging System (Analytik Jena, Germany) were employed to examine the electrochemiluminescence of indicated proteins.
5
Nrf2 and HO-1 Signaling Pathway Assay
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7,8-DHF (purity >99%) was procured from Sigma-Aldrich; Merck KGaA. Anti-Nrf2 (cat. no. BS1258) and anti-HO-1 (cat. no. BS6626) antibodies were supplied by Bioworld Technology at a dilution of 1:1,000. Anti-actin (cat. no. 4970) and anti-lamin B (cat. no. 13435) antibodies were obtained from Cell Signaling Technology, Inc. at a dilution of 1:5,000. Secondary HRP-conjugated goat anti-rabbit (cat. no. BS13278) antibody was purchased from Bioworld Technology, Inc. and used at a dilution of 1:5,000. The Cell Counting Kit-8 (CCK-8) and nuclear protein extraction kit were purchased from Beyotime Institute of Biotechnology. The superoxidase dismutase (SOD) and malondialdehyde (MDA) assay kits were supplied by Nanjing Jiancheng Bioengineering Institute. The fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) was obtained from Molecular Probes; Thermo Fisher Scientific, Inc.
6
Western Blot Analysis of Nuclear Proteins
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Nuclear proteins were extracted using a nuclear protein extraction kit according to the manufacturer’s instructions (Beyotime Co., Nantong, China). Protein concentrations were determined with a bicinchoninic acid protein assay kit (Beyotime). Aliquots of cell lysates containing 20 μg protein were separated in 10% polyacrylamide gels and electrophoretically transferred to PVDF membranes (Millipore) using a Bio-Rad criterion blotter. Membranes were blocked in 5% non-fat dried milk in TBST at room temperature for 1 h and then incubated with either anti-phospho-CREB (1:3000 dilution; Millipore) or anti-CREB (1:2000 dilution; Millipore) at 4 °C overnight. The blots were then washed and incubated with horseradish peroxidase-labeled secondary antibodies (Sigma) at 37 °C for 1 h. Immunoreactive bands were detected with enhanced chemiluminescence Western blotting detection reagents (Beyotime). The blots were exposed to X-ray film for radiography of the bands and were digitally detected and measured using a LAS3000 Bioimage Analyzer (Fuji Photo Film, Tokyo, Japan).
7
Nuclear Protein Extraction Protocol
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Nuclear extracts were prepared using a nuclear protein extraction kit (Beyotime, Shanghai, China) in accordance with the manufacturer’s instruction. Briefly, cells were rinsed twice with PBS and resuspended in reagent A to extract the cytoplasmic protein fraction. After extraction of cytoplasmic proteins, the pellets were resuspended in 50 µL nuclear protein extraction reagent. After agitation at 4 °C and centrifugation at 12,000×g for 10 min, the supernatant containing the nuclear proteins was harvested. Nuclear extracts were used for further analysis or stored at − 80 °C.
8
Quantitative Analysis of Notch2 and Hey1 Proteins
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Cells were washed 3 times with pre-cooled phosphate buffered saline (PBS) at 4°C. Total proteins were isolated by using cell lysates and quantified by BCA methods. In addition, nucleus proteins were also determined by BCA methods after being obtained using nuclear protein extraction kit (Beyotime, Jiangsu, China). After separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transfer onto PVDF membranes and incubated with 50 μg/L skim milk powder for 1 hour. TBST solution were used to wash membranes 3 times. Rabbit anti-mouse NOTCH2 and HEY1 primary antibodies (Abcam, USA, 1: 1000) were subsequently added to membranes, respectively. After 12 hours incubation at 4°C, all membranes were subjected to 3 times washing with TBST solution and goat anti-rabbit anti-IgG antibodies (1: 2000, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., China) were used to incubate membranes for 2 hours at room temperature. Three times washing with TBST were carried out once again. Finally, the blots were visualized by enhanced chemiluminescence Plus and integrate optical density was measure through software Lab Works 4.5. In this research, GAPDH was served as internal reference of total NOTCH2 and HEY1 proteins, while Histone H3 was set as the internal reference of nucleus NOTCH2 protein.
9
Affinity Purification of Nuclear Protein Complexes
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We linked three labels (FLAG-DDX18, HA-Drosha, His-DGCR8) to the indicated proteins. The prepared cells transfected with FLAG-DDX18, HA-Drosha, and His-DGCR8 plasmids were collected for nuclear protein extraction followed by coimmunoprecipitation. A nuclear protein extraction kit (P0027, Beyotime) was used according to the manufacturer’s protocol. In brief, alternate vortexing and centrifugation combined with the extraction kit were used to separate the total proteins into nuclear and cytoplasmic proteins. At the same time as the cell nuclear proteins were prepared, protein A/G magnetic beads (B23201; Bimake, Shanghai, China) were preincubated on a spinning wheel at 4°C for 30–60 minutes and washed three times with PBS. The antibody complex was then suspended in the nuclear protein solution. After the protein solution was fully combined with the magnetic bead–antibody complex, the extraction buffer was washed three times. Magnetic separation was performed by heating. The immunoprecipitate was collected and western blotting was performed.
10
Nuclear Protein Extraction Protocol
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Nuclear protein was extracted using a nuclear protein extraction kit (Beyotime, China, P0028) according to the manufacturer's protocol. The human lamin B and β-actin antibodies were used as the internal controls for protein loading, and relative expression levels were quantified using Quantity One software.
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