Thermo Fisher Scientific
NE-PER™ Nuclear and Cytoplasmic Extraction Reagent Kit
78833
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The WNT pathway is essential for embryonic development and adult tissue homeostasis. Deregulation of this pathway drives cancers of the stomach, intestine, and breast, among others. I am interested in studying the molecular mechanisms of oncogenic WNT signal transduction to identify potential targets for therapeutic intervention in colon cancer. The goal of this experiment is to optimize the nuclear-cytoplasmic fractionation in HAP1 cells using this kit.
Protein Biochemistry
HAP1 cell pellets
Fractionation should be fresh samples
Cells were trypsinized and washed twice with 1× PBS. Nuclear–cytoplasmic fractionation was performed according to the manufacturer's instructions. Equal volumes of each fraction were analyzed across treatment groups. Fraction purity was confirmed by immunoblotting for α-tubulin (cytoplasmic marker) and BCL9 (enriched in the nuclear fraction). Enrichment of β-catenin in the nuclear fraction following WNT ligand treatment is consistent with previous reports.
Fast and convenient
None
Fractionation purity at >90% in less than 2 hours.