Alex Palazzo is an Assistant Professor in the Department of Biochemistry at The University of Toronto.
Yesterday I gave a talk. Everything was fine although I thought I was a little wordy.
So instead of writing something I'll throw you a few pictures.
Here is a picture of a two cells that were microinjected with mRNA. The mRNA encodes a protein that is cotranslationally inserted into the endoplasmic reticulum (ER), and thus the mRNA itself is targeted to this same intracellular location. The cells were fixed 2hrs post-injection and then stained using a fluorescent probe the hybridizes against the exogenous transcript. This technique of labelling mRNA is called fluorescence in situ hybridization, or FISH.
When the mRNA is translated, the newly synthesized protein can be detected in the fixed cells by immunofluorescence. In this technique a fluorescent antibody that binds to your protein of interest, is employed as the probe.
To demonstrate that microinjected mRNA does result in the production of an ER targeted protein I took various pictures of the same field of cells. The image on the left is the injection marker and indicates which cell was microinjected. Note that the cell's nucleus lights up, this is because I microinjected nuclei and the marker is too large to cross nuclear pores. The second image is of the newly synthesized protein, the third image is of the a general ER marker (in this case Trap-alpha) and the fourth image is a color overlay of image two (green) and three (red).
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