I used 0.5 ug plasmid. Cell confluency around 60%. Transfection time is 48 hour. I used empty plasmid (pCMV-3Tag-4A) and only Lipofectamine as a negative control. However, I used EGFP plasmid tag for positive control. I found Myc tag plasmid transfection rate is only 5 to 10% where EGFP tag plasmid transfection rate is 60 to 70%. I am wondering why it low ? Can any one explain vector pCMV-3Tag-4A and pEGFP-C1 both have tag but myc tag did not express but EGFP is expressed. Is it functional and non functional peptide ?
Hi Nazmul Islam
The Myc tag plasmid may have a less active promoter in WT HeLa cells compared to the EGFP tag plasmid, resulting in lower expression levels.
Optimizing the transfection protocol by testing different reagents, adjusting plasmid amount, exploring alternative promoters, and trying different cell confluency or transfection times could improve your transfection efficiency.
Both Myc and EGFP tags are functional peptides, but their expression depends on the promoter used. So, for Myc tag to be expressed, all these factors must be carefully considered and optimized to enhance transfection efficiency in your cells.
Marzieh Rostaminejad Thank you. pCMV-3Tag-4A and pEGFP-C1 for this vector which one will be expressed ? I saw the gene map but I am not sure which one is functional ? Can you explain it.
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