Myocardial proteins were extracted using the Tissue Total Protein Extraction kit (Beyotime Institute of Biotechnology, Jiangsu, China). Nuclear proteins were isolated using a Nuclear Extraction kit (Beyotime Institute of Biotechnology) in accordance with the manufacturer's instructions. The protein concentration was quantified by a Bradford protein assay (Beyotime Institute of Biotechnology). Equal amounts of protein (40 µg from each group) were loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel, and then transferred onto a polyvinylidene fluoride membrane. Following blocking for 1 h with 5% skimmed milk at room temperature, the membranes were incubated with the primary antibody for HMGB1 (rabbit polyclonal antibody; diluted 1 µg/ml (1:1,000); cat. no. ab191583), RAGE (rabbit polyclonal antibody; 1:1,000; cat. no. ab37647), TLR-2 (rabbit monoclonal antibody; 1:5,000; cat. no. ab108998), TLR-4 (mouse monoclonal antibody; 1:5,000; cat. no. ab30667) and β-actin (rabbit polyclonal antibody; 1:1,000; cat. no. ab1801) overnight at 4°C, followed by incubation with the corresponding horseradish-conjugated secondary antibody (goat anti-rabbit; 1:10,000; cat. no. ab175773 or goat anti-mouse; 1:10,000; cat. no. ab97040) for 1 h at room temperature. The protein bands were quantified using scanning densitometry, and the results were normalized to the expression of β-actin.
Nuclear extraction kit
Manufactured by Beyotime
Sourced in China
The Nuclear Extraction Kit is designed for the rapid and efficient extraction of nuclear proteins from various cell types. It utilizes a carefully optimized buffer system to selectively isolate nuclear fractions, enabling the study of nuclear-localized proteins and their regulatory mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Transam nf κb p65 colorimetric kits, by Active Motif (2 mentions)
Microplate reader, by Agilent Technologies (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ecl western blot kit, by Thermo Fisher Scientific (2 mentions)
Bradford protein assay, by Beyotime (1 mentions)
Total tissue protein extraction kit, by Beyotime (1 mentions)
Ab13370, by Cell Signaling Technology (1 mentions)
Chemiluminescence, by Merck Group (1 mentions)
D5s6h, by Cell Signaling Technology (1 mentions)
Emsa assay kit, by Beyotime (1 mentions)
Enhanced chemiluminescence, by PerkinElmer (1 mentions)
Microplate spectrofluorometer, by Agilent Technologies (1 mentions)
Ab133104, by Abcam (1 mentions)
Emsa kit, by Thermo Fisher Scientific (1 mentions)
Ab38155, by Abcam (1 mentions)
Chemiluminescence system, by Bio-Rad (1 mentions)
Ab13243, by Abcam (1 mentions)
34 protocols using nuclear extraction kit
1
Quantification of Myocardial Protein Expression
2
Overexpression of Sp1 and AhR Transcription Factors
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For overexpression experiments, COS-7 cells were transfected with the constructs of the Sp1 or AhR gene. The Nuclear Extraction Kit (Beyotime, Haimen, China) was used to extract overexpressed nuclear protein from COS7 cells. EMSA was performed using the EMSA Assay Kit (Beyotime, Haimen, China). The mixtures containing unlabeled probe, nuclear extract (4 μg) and binding buffer were preincubated at 23°C for 10 min. Next, 0.1 pmol of the biotinylated probe was added and incubated at 23°C for 20 min in a total volume of 9 μL. The mixture was subjected to electrophoresis in 4.5% non-denaturing polyacrylamide [acrylamide/bisacrylamide 29:1 (v/v)] gels, followed by the addition of bromophenol blue in a blank hole as an indicator. The gel was transferred to a nylon membrane (+) after the bromophenol blue appeared at approximately 2 cm from the bottom of the gel. The nylon membrane (+) was crosslinked for 2 min under UV light (254 nm). The nylon membrane (+) was then processed with the EMSA Assay Kit (Beyotime, Haimen, China), followed by the detection by chemiluminescence (Millipore, Bedford, MA, United States). For competition experiments, 100-fold molar excess of unlabeled probe was used. For supershift experiments, 1 μL of anti-Sp1 (Abcam, ab13370), and anti-AhR (Cell Signaling Technology, D5S6H) were used.
3
Subcellular Protein Extraction and Validation
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After treatment, according to the instructions provided by the manufacturer, collect cells and use the nuclear extraction kit (Beyotime, China) to extract the nucleus and cytoplasmic protein, and determine the quality and purity of subcellular classification by using the immunoblotting of antibodies against the cytoplasm (β-actin) and cytoplasmic (Histones H3) protein.
4
Fractionation and Analysis of circ-PRKCI
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RCC cells were first washed with pre-cooled PBS and then treated with a cell separation buffer and a cell fragmentation buffer. We then used the Nuclear Extraction Kit (Beyotime) to isolate the cytoplasmic and nuclear components. circ-PRKCI content in the cytoplasmic and nuclear components was detected using qRT-PCR.
5
Western Blot Analysis of Renal Proteins
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Cytoplasmic and nuclear proteins were extracted from frozen renal tissues with a nuclear extraction kit (cat no. P0027; Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions. GAPDH was used as internal control of cytoplasmic protein and Lamin B was used as an internal control of nuclear protein. A BCA protein assay kit was used to determine the protein concentration (cat no. P0012S; Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg) were separated by 12% SDS-PAGE at 100 V for 3 h. After electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes at 200 mA for 2 h. The transferred membranes were incubated overnight at 4°C with rabbit anti-mouse polyclonal antibodies for DJ-1 and Nrf2 (each at 1:800 dilution) in Tris-buffered saline containing Tween-20 (TBS-T) containing 5% skimmed milk. After washing three times in TBS-T, membranes were incubated with anti-rabbit immunoglobulin G conjugated to horseradish peroxidase at a dilution of 1:2,000 in TBS-T containing 5% skimmed milk for 2 h at room temperature. The immunoreactive bands were visualized by enhanced chemiluminescence (PerkinElmer, Inc., Waltham, MA, USA) and captured on X-ray film. The optical density of the bands was measured with a BandScan V4.0 imaging analysis system (http://bandscan.software.informer.com/ ).
6
EMSA for NF-κB DNA Binding Assay
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The principle of EMSA is that DNA-protein complexes migrate at a slower rate than unbound DNA in a gel. The DNA binding activity of NF-κB was determined using the EMSA kit (Viagene Biotech, Ningbo, China), according to the manufacturer's protocol (22) . Briefly, nuclear protein was extracted using a nuclear extraction kit (Beyotime Institute of Biotechnology). Protein concentration was determined using the BCA method. The nuclear protein (15 µg) was mixed with a biotin-labeled NF-κB probe for 20 min at room temperature. Subsequently, the DNA-protein complex was separated using a 6.5% polyacrylamide gel at 180 V for 80 min, transferred onto a nylon membrane at 360 mA for 1 h and crosslinked to the nylon membrane under UV light for 30 min. The bands were visualized using streptavidin-HRP and an ECL kit. The sequence of the probe used in EMSA was 5'-AGT TGA GGG GAC TTT CCC AGG C-3'.
7
Quantifying Inflammatory Signaling Pathways
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Fluorescence assays were used to determine activities of MMP-3, MMP-2/9, COX-2, and NF-κB-p65. The cells seeded on 96 well/plates were treated with hypoxia with or without FGF21 for 8 h and then lysed with distilled water and then conducted according to the manufacturer’s instruction. To evaluate MMP-3 activity, the fluorescence was measured at 325/395 nm. To evaluate MMP-2/9 activity, the fluorescence was measured at 320/405 nm. To evaluate COX-2 activity, the fluorescence was measured at 535/587 nm.
Colorimetric assays were used to determine the transcriptional activity of NF-κB-p65 in nuclear extract. The cells seeded on 96 well/plates were treated with hypoxia with or without FGF21 for 8 h and then carried out with nuclear extraction kit (Beyotime, Haimen, China). The process was conducted according to the manufacturer’s instruction. The colorimetric readout at 450 nm was obtained. The transcriptional activity of NF-κB-p65 was calculated according to the standard curve.
Colorimetric assays were used to determine the transcriptional activity of NF-κB-p65 in nuclear extract. The cells seeded on 96 well/plates were treated with hypoxia with or without FGF21 for 8 h and then carried out with nuclear extraction kit (Beyotime, Haimen, China). The process was conducted according to the manufacturer’s instruction. The colorimetric readout at 450 nm was obtained. The transcriptional activity of NF-κB-p65 was calculated according to the standard curve.
8
Fractionation of Aortic Endothelial Proteins
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Nuclear and cytosolic fractions were prepared from freshly isolated aortic endothelium tissue or cells using a commercially available nuclear extraction kit (Beyotime Institute of Biotechnology, China). In all, ∼75 mg of aortic endothelium tissue was minced or cells were grown on 100 mm plates until 80–90% confluency, then homogenized in Cytoplasmic Extraction Reagent I buffer containing protease inhibitor cocktail Complete, EDTA-free. After a series of wash steps, nuclear proteins were extracted in high salt Nuclear Extraction Reagent buffer supplemented with protease inhibitors. The cytosolic fraction was spun at 100,000 g at 4 °C for 60 min to obtain a pure cytosolic fraction.
9
Quantifying Nuclear NF-κB Activity
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Nuclear extracts from hearts and HL-1 cells were obtained by a nuclear extraction kit (Beyotime Biotechnology, China) according to the manufacturer’s protocol. The DNA-binding activity of NF-κB p65 in nuclear extracts was analyzed by using TransAM™ NF-κB p65 Colorimetric kits (Active Motif, USA) in duplicate and detected at a wavelength of 450 nm by a microplate spectrofluorometer (Bio-Tek, USA) as described.
10
Quantifying HIF-1α DNA Binding Activity
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HIF-1α DNA binding activity was measured using a commercial assay kit (Abcam, ab133104). Briefly, nucleoprotein was extracted from HepG2 cells using a Nuclear Extraction Kit (Beyotime, China). Then, the obtained samples were added to the wells of transcription factor HIF-1α plate and incubated overnight at 4 °C. After incubation, diluted HIF-1α primary antibody was added to each well and incubated for 60 min at room temperature. Subsequently, diluted goat anti-rabbit HRP conjugate was added to each well and incubated for 60 min at room temperature. HIF-1α DNA binding activity were measured at 450 nm using a microplate reader (Biotek, USA).
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