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3x flag tagged cloning -> western -> multiple bands but not non-specific - Please help me!! (Apr/19/2010 )


I cloned my cDNA of interest into CMV-14 vector (3x flag, C-term tagging, Sigma)
I am trying to confirm the proper expression of my cloned cDNA in two ways. 1) immuno cytochemistry 2) western
My protein normally locates in the mitochondria, and I could detect it in mitochondria by immunocytochemistry.
But, in western blot, I detected 4 bands...I could not see these 4 bands in mock transfected or nothing transfected cells.
I sequenced the cDNA, and no error in PCR was made during the cloning step...
I wonder why 4 bands appeared in transfected lanes, and no non-specific bands in control lanes..
I heard sometimes the sigma flag antibody shows non-specific bands,
but in my case, those 4 bands were not detected in control lanes, which means it's not non-specific
Can someone please.. please.. help me??

The MTS signal locates at the very first region of the gene, that's right after the first methionine.
There are 4 more methionines after the first one, but.. I inserted Kozac (ACCATG) to ensure it starts from the first ATG.
Furthermore, I could detect the protein by cytochemistry, and it was in mitochondria, which means the translation really started from the beginning (including MTS which is just after the first ATG)

I will be so much grateful if you could help me!


Do you have cell death after this transfection? if so, then you better do one or two steps more of PBS washing before lysing your cells.