Protein delivery in HEK-293 using Magnetofection

Introduction of proteins into HEK-293 was performed using lipid-based transfection reagent and CombiMag from OZ Biosciences.

This paper described the use of lipid-based reagent and magnetic nanoparticles reagent (usually dedicated to nucleic acids transfection) for protein delivery. His-alpha-synuclein fibrils were incubated with Lipofectamine LTX anc CombiMag magnetic nanoparticles for 45 minutes before transfection onto a magnetic plate in FBS-free culture medium.

paper reference: PLoS One. 2012;7(12):e52868. doi: 10.1371/journal.pone.0052868.

p62/SQSTM1-Dependent Autophagy of Lewy Body-Like α-Synuclein Inclusions.
Watanabe Y, Tatebe H, Taguchi K, Endo Y, Tokuda T, Mizuno T, Nakagawa M, Tanaka M.

Abstract

α-Synuclein is the main component of Lewy bodies, the intraneuronal inclusion bodies characteristic of Parkinson's disease. Although α-synuclein accumulation is caused by inhibition of proteasome and autophagy-lysosome, the degradation of α-synuclein inclusions is still unknown. Formation of Lewy body-like inclusions can be replicated in cultured cells by introducing α-synuclein fibrils generated in vitro. We used this cell culture model to investigate the autophagy of α-synuclein inclusions and impaired mitochondria. The intracellular α-synuclein inclusions immediately underwent phosphorylation and ubiquitination. Simultaneously they were encircled by an adaptor protein p62/SQSTM1 and directed to the autophagy-lysosome pathway in HEK293 cell line. Most phospho-α-synuclein-positive inclusions were degraded in 24 h, however, lysosomal dysfunction with bafilomycin A1 significantly affected their clearance. Moreover, inhibition of autophagy by Atg-5 siRNA treatment reduced the incorporation of α-synuclein inclusions into LC3-positive autophagosomes. Knockdown experiments demonstrated the requirement of p62 for α-synuclein autophagy. These results demonstrate that α-synuclein inclusions are preferred targets for p62-dependent autophagy. Next, we investigated the autophagic clearance of impaired mitochondria in α-synuclein inclusion-containing cells. Impaired mitochondria were almost completely eliminated after mitochondrial uncoupling even in the presence of α-synuclein inclusions, suggesting that mitochondrial clearance is not prevented by α-synuclein inclusions in HEK293 cells.

CombiMag reagent is the only existing method based on the use of magnetic nanoparticles (Magnetofection™ ) , for improving your transfection reagent efficiency. It has been designed to be employed in association with any transfection reagent.

For Protein delivery  and for Antibody delivery into cells OZ Biosciences has also developped specific transfection reagent : Pro-DeliverIN and Ab-DeliverIN respectively.