I want to localize a protein in cyanobacterial Synechococcus PCC7942.The problems I met is that there was no fluorescence whether eyfp was in N- or C- terminal of my protein. The part of my protein in fusion protein, however, has its activity in vivo. The eyfp gene is okay because there was a strong bioluminescence observed in the positive strain.
Since the size of my protein is 82 kD, plus the eyfp, the fusion protein should has the size of 109 kD. Is there the possible that the protein is too large to be work out?
Can anyone give me any suggestions? Thanks very much!
What is the state of your protein in buffer? Is it monomer or will it construct into any multimer like dimer or hexamer? You said "fusion protein", I suppose you mean multimer, then the eyfp maybe hidden in the multimer. So you have to keep the protein in monomer state then check the flourescence. And I don't know the connection between the activity in vivo of your protein and the flourescence of the eyfp you put in N- or C- terminal.
One more thing is that is it possible that eyfp detached during the expression of your protein or purification of your protein, then you may need to do the SDS-page at every step.
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