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3x flag tagged cloning -> western -> multiple bands but not non-specific - Please help me!! (Apr/18/2010 )

Hello,

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!

-thedaywemet-

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.

-Curtis-