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Why does it take so long to see induced eukaryotic proteins?

Elongation Protein mRNA Rate

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#1 doxorubicin

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Posted 16 October 2011 - 12:57 AM

In our system, we potently induce endogenous mRNA transcription that we can detect at a maximal cytoplasmic level by 3 hours, but we do not see the proteins by Western blot until about 16 hours. I haven't found a good article, but online books (mostly textbooks) indicate that translation elongation occurs at a rate of 2 to 10 amino acids per second in eukaryotes, so even with the slower rate it would only take about 8 minutes to produce a 500-amino acid protein.

Just to make the calculation easy, let's assume that no translation occurs prior to 3 hours and that mRNA levels stay constant from 3 hours post-stimulation to 16 hours when the proteins are visible by Western. I think that would mean that each RNA will have to be translated at least 100 times before we see the protein. Now if you include the fact that each mRNA might be simulaneously translated by 4, 5, 6, 7, 8....ribosomes, then we're looking at maybe 300-500 proteins produced per mRNA required before we can see the protein by Western. Of course, some/most mRNAs will be degraded and replaced by new mRNAs.

Do these calculations sound reasonable?

I guess the best way to understand this is to calculate the detection limits of each antibody using recombinant protein and then determine how much total protein must be loaded per lane to observe the signal. This would allow quantitation of protein amounts, which when combined with good qPCR data would give some solid numbers.

Can anyone offer some more insight?





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