Nuclear extracts of the HEK-293 and H9c2 cells were prepared with an NE-PER® Nuclear Protein/Cytoplasmic Protein Extraction Kit (Thermo Fisher Scientific, Inc.). The protein concentration of the nuclear extract was determined using Bio-Rad Protein Assay Reagent and stored at –80°C until use. Biotinylated double-stranded oligonucleotides (30 bp) were used that contained SNP sites as probes. A LightShift® chemiluminescence electrophoretic mobility shift assay (EMSA) kit (Thermo Fisher Scientific, Inc.) was used for the DNA-protein binding reaction to explore the interaction between the DNA fragment of ATG16L1 gene promoter and the nucleoprotein. Unfortunately, due to the limitations of EMSA experimental technology, it is impossible to determine whether the protein directly interacts with the polymorphic sites on the promoter or indirectly affects the transcription process of the gene.
Ne per nuclear protein cytoplasmic protein extraction kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NE-PER® Nuclear Protein/Cytoplasmic Protein Extraction Kit is a laboratory product designed for the extraction and separation of nuclear and cytoplasmic proteins from cells. It provides a consistent and efficient method for the isolation of these protein fractions.
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2 protocols using ne per nuclear protein cytoplasmic protein extraction kit
1
Analyzing ATG16L1 gene promoter interactions
2
Subcellular Fractionation Using NE-PER Kit
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An NE-PER Nuclear Protein-Cytoplasmic Protein Extraction Kit (78833, Thermo Fisher, USA) was used to separate the subcellular fractions in accordance with the manufacturer's instructions. Brie y, BV2 cells were washed with ice-cold PBS and lysed in CER I working reagent protease containing protease inhibitors for 10 min on ice. After adding CER I working reagent, cytosolic protein isolation was performed by centrifuging the supernatant. Then, the addition of the NER working reagent to the cell pellet caused the release of the cytoplasmic content. The protein concentration was quanti ed using the BCA Protein Assay Kit (23225, ThermoFish, USA).
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