Plus,

When larger tag (such as BioID) is fused to the N-terminal of the protein, the fused truncated mutant protein could be detected on the western blot; however, I could not see any band when the same tag is fused to the C-terminal of the same mutant variant instead of the N-terminal...

If the allele contains a frame-shift and creates a truncated protein, then that means the sequence has a premature stop codon. I would not expect a C-terminal tag to be expressed since translation would end before the ribosome reached the sequence for the tag.

Hi there,

FS mutation might induce protein unstability because the sequence downstream the mutation will be disordered and therefore more prone to degradation.

Katie A Burnette

Hi Katie, thanks for the reply,

I have actually removed the stop codon when assembling the plasmid in which the Tagging proteins are fused to the C terminal of the POI, and checking through snapgene everything seems in-frame, and the stop codon that theoretically should stop everything locates after the Tagging protein.

Dominique Liger

Hi Dominique, thanks for the reply.

I have actually used a commercially available protease inhibitor cocktail when I was extracting proteins during cell lysis, any suggestions to further stabilize these unstable protein candidates?

Check for "non-standard" stop codons and transcriptional stalling. Both of those can lead to degradation. You might also be activating the nonsense mediated decay pathway.