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Timing of protein measurement in a translation-blocking experiment

This was written to a researcher in response to his question about timing of assays in a translation-blocking experiment.


Since you are using a translation blocker, the optimum delay between treatment and analysis will be strongly affected by the turnover time of the protein. It will also be affected by the delivery technique used to get the Morpholino into the cytosol. Rapid techniques like electroporation or microinjection will get the Morpholino in quickly and start the knockdown immediately, while endocytosis-mediated techniques like Endo-Porter or Vivo-Morpholinos might take a day to achieve sufficient cytosolic Morpholino concentrations for a good knockdown. Once translation is suppressed, the preexisting protein is degrading without replacement. However, some proteins are very stable and might have half-lives of days. Rapid-turnover proteins like most transcription factors will disappear quickly, perhaps in hours.

If you are targeting cells in culture using an unmodified Morpholino with Endo-Porter as an endosomal escape agent, and you are targeting a protein with a one-day half-life, I'd try about three days between oligo delivery and measurement of the protein. That gives a day for delivery and two half-lives for protein degradation.

If the protein is especially stable, it might take another delivery of Morpholino on day four to keep translation suppressed long enough for most of the protein to degrade.

I think you can tell by my tone that all of these estimates are pretty rough. Still, if you consider delivery and half-life I think you can find an effective time course within a few experiments.

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