The treated SH-SY5Y cells were lysed to extract the proteins using RIPA lysis and extraction buffer (Thermo Fisher). Nuclear proteins were extracted using Cytoplasmic And Nuclear Protein Extraction Kit (BosterBio, USA). 5 µg of total protein was loaded into 8% of sodium dodecyl sulphate-polyacrylamide gel and separated by electrophoresis. The separated protein was transferred from the gels to the PVDF membranes and the PVDF membranes were blocked by 5% of fat-free milk for 1 h at room temperature. The proper primary antibodies were used to probe the target proteins overnight at 4°C followed by incubating the membrane with proper secondary antibodies (ThermoFisher) for 2 h at room temperature. The strength of protein signal was detected using SignalFire™ ECL Reagent (CST, USA). The primary antibodies used in this study were: BACH1 (sc-271211, 1:1,000 dilution), NRF2 (ab137550, 1:1,000 dilution), Lamin B2 (ab233530, 1:800 dilution), and β-actin (ab8227, 1:5,000 dilution).
Nuclear and cytoplasmic protein extraction kit
Manufactured by Boster Bio
Sourced in China
The Nuclear and Cytoplasmic Protein Extraction Kit is a laboratory tool designed to separate and purify nuclear and cytoplasmic proteins from cells or tissues. It provides a method for the efficient extraction and isolation of these proteins for further analysis and study.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Solarbio (2 mentions)
Bca protein assay, by Beyotime (2 mentions)
Proper secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Bardford protein assay, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Biotin 3 end dna labeling kit, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Parp1, by Abcam (1 mentions)
Histone 3, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Horseradish peroxidase labeled secondary antibody, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Beyotime (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Novus Biologicals (1 mentions)
β actin, by Novus Biologicals (1 mentions)
Myd88, by Novus Biologicals (1 mentions)
Ripa lysate buffer, by Beyotime (1 mentions)
β actin 66009 1 ig, by Proteintech (1 mentions)
Total protein extraction reagent, by Boster Bio (1 mentions)
Mitochondrial membrane potential assay kit, by Beyotime (1 mentions)
12 protocols using nuclear and cytoplasmic protein extraction kit
1
Western Blot Analysis of Cellular Proteins
2
Plasmid Transfection and Protein Interaction Analysis
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Constructed plasmids encoding pcDNA-myocardin-Flag, pcDNA-SOX9-Myc, pcDNA-SRF were contransfected into HEK293T cell line (ATCC Cat#CRL_3216, RRID: CVCL_0063) or PI3Kγ-KD VSMCs. Cells were lysed 48 h post-transfection, nuclear protein fractions extracted by utilizing a cytoplasmic and nuclear protein extraction kit according to the manufacturer's protocol (Boster Biological Technology, China) were incubated with anti-Myc or anti-Flag or anti-SRF antibody overnight at 4 °C. Normal IgG was acted as a negative control. Protein A + G Agarose beads were added and slowly swung for 2 h at 4 °C.The immunoprecipitates were washed five times with pre-cooling PBS and then mixed with SDS-PAGE buffer, followed by western blotting analysis. For western blotting, whole cell lysates were prepared with lysis buffer containing protease inhibitors and quantified using the Bardford Protein Assay (Bio-Rad). Equal amounts of denatured whole cell lysates of VSMCs were separated by SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking, proteins were detected with corresponding primary antibodies and subsequently with appropriate HRP-conjugated secondary antibodies. Immunoreactivity was visualized with enhanced chemiluminescence detection reagents and recorded using ChemiDoc imaging system (Bio-Rad). Protein expression levels were normalized against β-actin.
3
Assessing NF-κB Binding to TNF-α Promoter
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The binding of nuclear NF-κB to the TNF-α promoter was assessed by EMSA. Nuclear extracts were prepared from lung tissue using Cytoplasmic and Nuclear Protein Extraction Kit (Wuhan Boster Bio-Engineering Limited), and centrifuged at 14,000 rpm for 10 min at 4°C. Supernatant was stored at -80°C until used for EMSA. NF-κB-specific oligonucleotide 5′- AGGGGGCTTTCCCT -3′ from the murine TNF-α gene promoter was synthesized (TaKaRa, Mountainview, CA, USA). DNA probes were labeled with biotin, using Biotin 3' End DNA Labeling Kit (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of nuclear protein were incubated with biotin-labeled NF-κB oligonucleotide probe for 30 min and run on 5% PAGE at 100 V. The gel and nylon membranes were layered as a sandwich in a clean electrophoretic transfer unit and transferred at 100V for 30 minutes. Transferred DNA was cross-linked to membranes with a hand-held UV (254 nm) for 5–10 minutes, and biotin-labeled NF-κB probe detected by chemiluminescence.
4
Analyzing NF-κB Pathway Activation
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Phosphorylated IKK and I-κB were by assessed by western blot analysis. Plasma extracts were prepared from lung tissue using Cytoplasmic and Nuclear Protein Extraction Kit (Wuhan Boster Bio-Engineering Limited), and centrifuged at 12,000g for 10 min. Protein extract concentration was estimated with a BCA Protein Assay kit (Beyotime Biotech). Equal amounts of protein (25 μg/lane) were resolved by 8% SDS-PAGE, and transferred onto a PVDF membrane. The membrane was then washed with TBST and blocked in TBST containing 5% non-fat dried milk, and further incubated with the respective specific antibodies to p-IKKα/β, IKKα, IKKβ, p-I-κB, I-κB, p-NF-κB p65 and NF-κB p65, and β-actin. Membranes were then incubated with appropriate HRP-conjugated secondary antibodies, and developed in ECL Western detection reagents (Millipore, Billerica, MA, USA). All experiments were repeated three times.
5
Subcellular Protein Extraction from Liver
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The cytoplasmic and nuclear protein was collected using nuclear and cytoplasmic protein extraction kit (Boster, AR0106). The liver was cut into small pieces and made to be tissue homogenate with ice-cold buffer A. Buffer B was added to the samples followed by 1 min centrifugation. The cytoplasmic supernatant was then collected. 100 mL ice-cold buffer C was added to centrifugal precipitate which containing 1 mL DTT and 5 mL 100 mM PMSF per mL Buffer C, 5 μL protease inhibitor. Then the supernatant was centrifuged at 4°C for 16 000 g, 10 minutes, and transferred to ice-cold clean micro-centrifugal tube as soon as possible to obtain nucleoprotein. Protein levels in the cytoplasmic and nuclear extracts were detected following the western blot procedure.
6
Cellular Fractionation Protocol
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Cellular cytoplasmic and nuclear fractions were separated using the Nuclear and Cytoplasmic Protein Extraction Kit (16B01A06; BOSTER).
7
Cellular Protein Extraction and Western Blot Analysis
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To obtain the nuclear and cytoplasmic protein of cells, the Nuclear and Cytoplasmic Protein Extraction Kit (Bosterbio, Wuhan, China) was used according to the manufacturer’s instructions. Protein concentrations were determined using BCA protein assays (Beyotime, Beijing, China). The protein was separated by 12 % SDS-PAGE gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane by electrolysis. After being blocked with 5% BSA in TBST for 1 hour at room temperature, the membranes were incubated with the primary antibodies overnight at 4° C. Here the following specific primary antibodies were used as follows: PARP1 (1:2000 dilution; Abcam, MA, USA), AIF (1:1000 dilution, Abcam, MA, USA), MIF (1:1000 dilution, Abcam, MA, USA), Histone-3 (1:500 dilution, Abcam, MA, USA), and GAPDH (1:1000 dilution, Abcam, MA, USA). Then the membranes were washed three times with TBST, and they were incubated with the corresponding secondary antibodies conjugated with horseradish peroxidase for 1 hour. The membranes were washed three times with TBST for 15 minutes each time. Finally, the immune response zones were detected using an ECL detection system (SmartChemi 420, Beijing, China). The results were analyzed by Image J software.
8
Protein Isolation and Western Blot Analysis
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Total protein was isolated with radioimmunoprecipitation buffer and then quantified using the BCA protein assay kit (Solarbio), as per the manufacturer’s instructions. Cytoplasmic and nuclear proteins were isolated using the Nuclear and Cytoplasmic Protein Extraction Kit (Boster, Beijing, China) according to the manufacturer’s recommendations. After sodium lauryl sulfate-polyacrylamide gel electrophoresis, the isolated proteins were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk and then blotted with the following antibodies from Abcam (Cambridge, MA, United States) or Cell Signaling Technology (Beverly, MA, United States): anti-phosphorylated (p)-mammalian sterile 20-like protein kinase 1 (MST1) antibody (1:1,000), anti-p-large tumor suppressor 1 (LATS1) antibody (1:1,000), anti-p-YAP antibody (1:2,000), anti-YAP antibody (1:1,000), anti-H3 antibody (1:1,000), and anti-GAPDH antibody (1:3,000) at 4 °C overnight. GAPDH and histone H3 were used internal loading controls. Following 1 h of incubation with horseradish peroxidase-labeled secondary antibody (1:2,000; Abcam), blots were developed with an enhanced chemiluminescence kit (Beyotime) as per the manufacturer’s recommendations.
9
Protein Expression Analysis in HT22 Cells
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After treatment, total protein from HT22 cells were extracted by using RIPA lysate buffer from Beyotime, and then tested for protein concentration using the BCA protein assay kit from Solarbio. Nuclear and Cytoplasmic Protein Extraction Kit (Boster, Beijing, China) was utilized to isolate the nuclear and cytoplasmic proteins from HT22 cells following the manufacturer’s recommendation. After sodium dodecyl sulphate polyacrylamide gel electrophoresis, the separated protein was transferred onto polyvinylidene fluoride membranes. Subsequently, the members were blocked for 1 h with 5% non-fat milk, and then probed at 4°C overnight with the special primary antibodies against TNIP2 (Novus, Shanghai, China), toll-like receptor 4 (TLR4; Novus), myeloid differentiation factor 88 (MyD88; Novus), NF-кB (Novus), and β-actin (Novus). The membranes were rinsed with TBST for 3 times and then immunoblotted for 1 h with horseradish peroxidase-conjugated secondary antibody (Novus) at 37°C. The immunoblots were visualized using the chemiluminescence reagent from Solarbio, following the product manual.
10
Antioxidant and Hepatoprotective Effects
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Puerarin (98% purity; Product code: S30646) and acetaminophen (99% purity; Product code: S31044) were purchased from Shyuanye (Shanghai, China). Superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), glutathione (GSH), reactive oxygen (ROS), aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (AKP), γ‐glutamyltransferase (γ‐GT), total bilirubin (TBIL) assay kits were purchased from Nanjing Jiancheng (Nanjing, China). Mitochondrial membrane potential assay kit was purchased from Beyotime (Beijing, China). Total protein extraction reagent and Nuclear and Cytoplasmic Protein Extraction Kit were purchased from Boster (Wuhan, China). The antibodies against Keap1 (10503‐2‐AP), Nrf2 (16396‐1‐AP), GCLC (12601‐1‐AP), GCLM (14241‐1‐AP), HO‐1 (10701‐1‐AP), NQO1 (67240‐1‐Ig) and β‐actin (66009‐1‐Ig) were obtained from Proteintech (Wuhan, China). The antibody against Lamin B (#13435) was obtained from Cell Signaling Technology (Danvers, MA, USA). The secondary antibody was obtained from Boster (Wuhan, China).
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