problem with overexpression - mRNA vs protein level (Aug/02/2007 )

I tried to overexpress a protein, and observed an increase in the mRNA level by RT-PCR, but cannot detect an increase in protein by western blot. Anyone has similar experience? Is it due to degradation of the protein or some regulatory mechanism at the translation level?

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Could be either of those issues. Are you sure the mRNA is being translated at all? There are many example of genes who's mRNAs are expressed, but the transcripts are processed and then store in p-Bodies, maternal granules, neuronal granules, etc instead of making all the way into a polysome for translation. Is it an endogenously expressed gene, or one from a plasmid? Does the overexpressed mRNA have the same 5' or 3' UTRs that the natural gene has? An artificial 5'UTR could lead to a total absence of protein expression if you are careful. Also, is it possible that your westerns are simply being done at saturation so any change in protein is being masked? If so, try exposing for less time or running less sample on each lane of your gel.

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You could also test if it is, in fact, a degredation issue by treating your cells with a proteosome inhibitor such as MG132. It doesn't really solve the problem but it will help you determine where the problem is.

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