I am trying to see the sub-location of my protein. I use E.coli-Pseudomonas shuttle vector, pUCP20, to construct GFP-protein fusion plasmid, which could be induced by1 mM IPTG. When I used Leica SP2 confocal microscopy, I can see the fluorescence. However, the SP2 could not get sharp image, which might be due to fainter fluorescence.
I know that it is easier to observe GFP in E. coli. But it is not often to see relative report in Pseudomonas. Does anybody have experience about this? which vector you use?
much appreciation for your valuable suggestions.
Does UV spectroscopy require longer wavelength radiation than IR spectroscopy?
I have experience of using pMRP9-1 EGFP plasmid with Pseudomonas i see the fluorescence very nicely... and it harbors the vector for quite a long time...
check maybe your confocol has some technical issues to deal with...
Hi Chen,
To see the expression of GFP in Pseudomonas is OK, to see protein location, it depends on the level of expression. Additionally, Pseudomonas aeruginosa has very strong autoflorescence that overlap with the GFP Ex/Em spectrum. If the expression is high this in not a problem, you can adjust the gain and intensity of excitation, but if it is middle low, I would change to something different like YFP or mCherry. Be careful with some ds-Reds because they are not monomers that is better for the fusion. I hope this helps.
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