Hello,
I hope you can give me some advice on this issue.
We have a tagged plasmid (pcDNA3.1+) where the protein of interest is tagged at both N-terminus (Myc) and C-terminus (Flag). No spacer sequences have been used.
When overexpressed in HEK293 cells, the plasmid is transcribed normally (confirmed with qPCR) and the protein can be visualised with a Flag WB (and is of the expected size). It can also be visualised using an antibody recognising the protein of interest itself. However, when using a Myc antibody, the blot is completely negative. The antibody works well as the positive control (another Myc-tagged protein) can be visualised perfectly.
I have checked and the Myc tag sequence is definitely in-frame so I don't know what the problem is. We have also tried deleting the ATG codon from the ORF thinking that perhaps translation was skipping the Myc tag but this made no difference.
Has anyone encountered similar issues and can give some advice? I am thinking to replace the Myc tag with Ha (I really need the N-terminal tag to work) but I am afraid I will encounter the same problem...
Thank you in advance!
A few ideas:
1. Could the Myc tag be hidden inside the protein?
Do you have a structure (try Alpha fold?) Is the N-terminus folded in? Even under denaturing conditions there might be some residual folding.
2. Could it be that the protein is being processed and the N terminal removed (i.e. Preprotein)? This is a common situation. You can try running https://services.healthtech.dtu.dk/services/ProP-1.0/ or similar
Good luck
Yoram
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