Membrane proteins were extracted from PMs using a cell membrane protein extraction kit (Thermo Fisher Scientific, Waltham, MA, USA). Cytoplasmic and nuclear proteins from PMs were extracted using a nucleoprotein Extraction Kit (Solarbio).
Nucleoprotein extraction kit
Manufactured by Solarbio
Sourced in China
The Nucleoprotein Extraction Kit is a laboratory tool designed to isolate and purify nucleoproteins from biological samples. It provides a reliable and efficient method for the extraction of these important macromolecular complexes.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti tlr4, by Santa Cruz Biotechnology (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Ripa lysate buffer, by Solarbio (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Solarbio (1 mentions)
Penicillin streptomycin mixture, by Solarbio (1 mentions)
Protease phosphatase inhibitor, by Solarbio (1 mentions)
Bca protein quantitative kit, by Solarbio (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Gapdh, by Proteintech (1 mentions)
7 protocols using nucleoprotein extraction kit
1
Membrane Protein Extraction Protocol
2
Western Blot Analysis of NF-κB Pathway
3
Anti-inflammatory Mechanisms of LPS-induced RAW264.7 Cells
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The following reagents were used in this research: RAW264.7 cells were purchased from the Typical Culture Preservation Committee Cell Bank, Chinese Academy of Sciences. DMEM (Solarbio, Beijing, China). FBS (Gibco, GrandIsland, USA). Penicillin-streptomycin mixture, Nucleoprotein extraction kit, BCA protein quantitative kit and protease phosphatase inhibitor (SolarBio, Beijing, China). LPS (Sigma-Aldrich, St. Louis, MO, USA). NO kit and ROS kit (Beyotime Biotechnology Shanghai, China). Mouse TNF-α Enzyme-linked immunosorbent assay (ELISA) kit and mouse IL-6 ELISA kit (4A Biotech Co, Ltd. Beijing, China). Evo M-MLV RT Kit with gDNA Clean and SYBG Green Premix Pro Taq HS qPCR kit (Tli RNadeH Plus, Accurate Biotechnology, Hunan, China). Antibody iNOS, COX-2, NF-κB P65, p-NF-κB P65 (Cell Signaling, USA). Primers were designed and synthesized by Thermo Fisher Scientific (Shanghai, China). The primers were shown in Supplementary Table 1 .
4
Protein Extraction from Testis Tissues
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A previously established method was employed to extract total protein from testis tissues (Liu et al., 2022a ). Testicular tissues were homogenized in a homogenizer after the addition of protein lysate. Following lysis at 4°C for 30 min, the mixture was centrifuged and the supernatant was aspirated as a total protein sample. Additionally, nuclear proteins were extracted using a Nucleoprotein Extraction Kit (Solarbio Biotechnology, Beijing, China). The protein concentration was then determined using the BCA protein assay kit (Solarbio Biotechnology, Beijing, China). The volume of the sample was determined based on the total amount of protein. Finally, the protein samples underwent electrophoresis and western blot analysis. The primary antibodies were GPX4 (1:1,000), FTH1 (1:2000), SCL7A11 (1:1,000), Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4 ) (1:1,000), TFR1 (1:1,000), FPN1 (1:1,000), Prostaglandin-Endoperoxide Synthase 2 (PTGS2 ) (1:1,000), cleaved-caspase-3 (1:500), Nrf2 (1:500) and GAPDH (1:5,000). GPX4, FTH1, SCL7A11, ACSL4, TFR1, and FPN1 came from Abmart (China). PTGS2, cleaved-caspase-3 and Nrf2 were sourced from Wanleibio (China). GAPDH was obtained from Proteintech (China). The GAPDH band was utilized for standardized quantitative analysis.
5
Nucleoprotein Extraction from HUVEC Cells
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HUVEC cell nuclei and cytoplasm were extracted with a nucleoprotein extraction kit (Solarbio Technology Co., Ltd., Beijing, China). Briefly, HUVECs were collected, and to facilitate cell lysis, the cells were thoroughly mixed with the cytoplasmic extraction reagent and allowed to stand at 4°C for 10 min. The supernatant was the cytoplasmic extract. The precipitate was mixed well with the nuclear extraction reagent and incubated at 4°C for 15 min. The supernatant after centrifugation was the nuclear extract. The protein concentration of the cytoplasmic and nuclear extracts was determined with s bicinchoninic acid (BCA) assay kit.
6
Western Blot Protein Extraction and Analysis
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Western blot assay After 48h of cell culture in each group, the medium was discarded, the cells were rinsed three times with PBS. Total protein, cytoplasmatic protein and nuclear protein were extracted according to the procedures of NucleoProtein Extraction Kit (Solarbio, China). The extracted proteins were quantified with BCA and added into SDS-PAGE gel electrophoresis system, after which the proteins in the gel were transferred to PVDF membranes and blocked in 10% BSA. Next, PVDF membranes were incubated overnight at 4 with primary antibodies, washed three times with TBST and incubated with HRP-labeled Goat Anti-Rabbit IgG for 2h at room temperature. They were visualized by enhanced chemiluminescence, photographed directly and quantified.
7
Studying Protein-AgNP Interactions
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MEL cells were induced with DMSO for 72 h, and then nuclear extracts were prepared using a nucleoprotein extraction kit (Solarbio, China). The nuclear extracts were added into the binding buffer with a final volume of 400 mL, as previously described [26] . Subsequently, AgNPs (1, 8 mg/mL) or Ag ions (0.8 mg/mL) were added and incubated at 30 C for 2 h. After the incubation, the complexes were centrifuged at 17,000 g at 4 C for 1 h. The pellets were washed twice with ice-cold PBS, and the bound proteins were dissolved in 20 mL of denaturing buffer (100 mM DTT, 5% SDS), followed by Western blotting.
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