Isolation of Highly Enriched Nuclear Proteome Using Optimized Methods from Neuroblastoma Cells

Yıl 2024, Cilt: 7 Sayı: 1, 38 - 51, 29.02.2024

Ayimgül Uzunyol , Murat Kasap , Gürler Akpınar

Öz

Objective Nuclei sits at the center of cells and orchestrates many cellular metabolisim. However, in proteomic studies, we lack of profound understanding of many processes regarding to nuclei due to the poor enrichment of its proteome. In this study, to understand this important organelle in detail, comprehensive evaluation of four different nuclear protein enrichment methods were conducted by using neuroblastoma cell lines (SH-SY5Y) as a model. Methods Nuclear proteins (NPs) have been isolated using either commercially available kits or density gradient centrifugation. The purity of the isolated nuclear proteins have been verified using Western Blot (WB) analysis with antibodies against histonH3, LaminA/C, GAPDH and Cyclophilin A. Further analysis have been performed by using 2-DE (2 dimentioanl gel electrophoresis) gels and the proteins have been identified by MALDI-TOF/TOF analysis.
Results: Results from this comparison study demonstrated that Q-Proteome nuclear protein isolation kit (Qiagen, USA) was superior when compared to other most commonly used differential and density gradient centrifugation nuclear protein enrichment methods. Collected fractions using this method gave bands only with anti-histone H3 and anti- LaminA/C antibodies but not with anti-GAPDH and anti-cyclophilin A antibodies which indicating the presence of nuclear protein enrichment. Approximately 70% of the proteins on 2-DE gels were resident nuclear proteins or predicted to be nuclear-associated.
Conclusion: Overall, we demonstrated that although it is not possible to obtain purified nuclear protein fractions, it is feasible to obtain highly enriched nuclear fractions from cells grown in culture. This new method which provided a better lysis and separation may allow comparative nuclear proteome analysis studies with high nuclear protein coverage. Our study provides comprehensive new insight into the biology of the nucleus and will serve as an important platform for nuclear protein isolation.

Anahtar Kelimeler

Nucleus, Proteomics, neuroblastoma cells, enrichment methods

Destekleyen Kurum

Tübitak

Proje Numarası

1002 Projesi, Proje No: 216S177

Kaynakça

• P KTwJs. Nuclear transport and cancer: from mechanism to intervention. Nat Rev Cancer. 2004;4(2):2004 4:2. doi:10.1038/nrc1274
• Laurila K, Vihinen M. Prediction of disease-related mutations affecting protein localization. BMC Genomics. 2009;10. doi:10.1186/1471-2164-10-122