Citation
- Authors: Sakuma C. et al.
- Year: 2022
- Journal: Biotechniques
- Applications: in vitro / DNA / FectoPRO
- Cell types:
- Name: Expi293F
Description: Human embryonic kidney Fibroblast
Known as: Expi 293-F, Expi, HEK-293 Expi - Name: HEK-293 6E
Description: Human embryonic kidney Fibroblast cell line genetically modified with a truncated version of EBNA1 which grows in suspension and chemically defined serum-free medium.
- Name: Expi293F
Method
Expi293 cells were transfected with plasmid pTT5 o/GFP (the National Research Council of Canada, Montreal, Canada). HEK293-6E cells (NRC) were transfected with plasmid pTT5-human PLXDC2 genes (UniProt ID Q6UX71, MW: 59,583, pI: 5.99) as previously described [5,14]. The transfected cells were grown in HEK293 expression medium (Kyokuto Pharmaceutical Industrial, Tokyo, Japan) on a shaker running at 135 rpm in a humidified 37◦C incubator in the presence of 5% CO2. Transfection of HEK293 cells in suspension was performed according to the published guideline described in detail [14,15] using plasmids and FectoPro (Polyplus transfection, Illkirch, France).
Abstract
We have developed a new Western blotting method of native proteins from agarose-based gel electrophoresis using a buffer at pH 6.1 containing basic histidine and acidic 2-(N-morpholino)ethanesulfonic acid. This gel electrophoresis successfully provided native structures for a variety of proteins and macromolecular complexes. This paper is focused on the Western blotting of native protein bands separated on agarose gels. Two blotting methods from agarose gel to PVDF membrane are introduced here, one by contact (diffusion) blotting and another by electroblotting after pre-treating the agarose gels with SDS. The contact blotting resulted in the transfer of native GFP, native human plexin domain containing protein 2 (PLXDC2) and native SARS-CoV-2 spike protein, which were detected by conformation-specific antibodies generated in-house.