[Transfection].
Transfection is a method to introduce DNA into mammalian cells. Calcium phosphate-mediated transfection is well used as one of the methods for transfection. The basic protocol is simple and does not require the elaborated instruments. It has been applied to introduce the heterogeneous DNA for integration into and expression from the chromosome. The proteins so expressed can be further analysed for their function and structure. In this section, a protocol for high efficiency transfection with calcium phosphate and its applications to membrane proteins are introduced. By exploiting the various transfection methods as tools for analyses such as chimeric gene expression and the site-directed mutagenesis, in the Na+, K+ -ATPase the N-terminus of alpha-subunit was shown to be a Na+ sensor, the ouabain binding site was found between the amino acid residues 69 and 200, and the assembly site of alpha- and beta-subunits was located within the 26 residues. In the case of Ca+ -ATPase, the newly isolated isoform was revealed to possess the same ATP-affinity as the already known ones, and the calmodulin-binding site was also shown to interact with the Na+, K+ -ATPase alpha-subunit.
Ohta
T
T
Basic Medical Sciences, University of Tsukuba, Japan.
D000818:Animals; D000252:Calcium-Transporting ATPases; D002478:Cells, Cultured; D004247:DNA; D005822:Genetic Vectors; D017506:H(+)-K(+)-Exchanging ATPase; D008565:Membrane Proteins; D000254:Sodium-Potassium-Exchanging ATPase; D014162:Transfection
D008565:Membrane Proteins; D004247:DNA; D017506:H(+)-K(+)-Exchanging ATPase; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase