my protein was not expressed on the eukaryotic expression plasmid in vitro？
I have encountered several proteins can not expressed when the gene were inserted into a eukaryotic expression plasmid and transfected into cells.
for example , In order to investigate the biological characteristic of feline PKR( Interferon-induced, double-stranded RNA-activated protein kinase ,PKR), the gene(CDS) of PKR were cloned from feline kidney cells，and inserted into p3Xflag-cmv-10 , pcmv-myc vector, then the recombinant plasmid were transfected into feline kidney cell F81，and the expression of PKR were detected by western blotting method, while the Tag protein can not be detected by the Corresponding tag antibody. The Recombinant plasmid were corrected and Confirmed by my several Colleagues. So where is the problem
Actually, many correct ORF Or CDS can express well,whether the protein is a cell or virus protein,while when they were cloned into a expression plasmid ,nothing could be detected by western blotting method nor could be detected by other method.
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