I am working on a co-localization experiment. I have shown that my protein of interest interacts with ATP5A, an inner mitochondrial membrane protein, by co-IP experiment. Now I want to do con-focal imaging to demonstrate the presence of my protein of interest in mitochondria. For that, I have cloned the ATP5A with dsRed Tag. I am getting very weak signal for DsRed Tagged ATP5A (signal is good for DsRed control) under fluorescent microscope in transfected cells. Has anyone experienced such problem or is there any suggestion to get better signal?
If you just want to show the co-loclization of your protein of interest with the mitochondria you might as well just stain the mitochondria with mitotracker. It could be that a tag interferes with Atp5 function and tagged version is expressed to a lower level, or tag is cleaved.
Thanks maria..
I have done the experiment with mitotracker. Now I want to provide strength to my data by demonstrating the co-localization with the the interacting mitochondrial protein (DsRed-ATP5A).
Do you know how much of your DsRed-tagged ATP5A is being expressed in your transfected cells? One potentially critical reason for a low DsRed signal is low expression of the transgene. Another possibility is that ATP5A levels may be very tightly regulated by the cell and any excess DsRed-ATP5A is broken down. Another possible reason is that the DsRed tag interferes with the synthesis, trafficking, or stability of the ATP5A. Although fluorescent protein tags often have little or no effect on the protein they are attached to, this is not always the case. The DsRed tag itself may be disrupting the protein at many levels from synthesis, folding, trafficking, and molecular interactions.
Which promoter have you used for the cloning of DsRed-ATP5A?. I think there are two possibilities: level of expression or protein stability. Maybe is a problem of expression and not of stability in this case. DsRed is one of the most stable fluorescent protein, and you can see normally in vacuoles (the cell try to degrade the protein) when you make fluorescent cell imaging, so I think it is not the problem.
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