[Arabidopsis] Replies about the faint fusion GFP

Guan Yue-Feng via arab-gen%40net.bio.net (by guanyf from gmail.com)
Sat Jan 16 19:49:27 EST 2010


Dear all,

For my previous post about the faint fusion GFP, I got man replies.
Here I post some of the suggestions that might also be helpful to 
others who is suffering similar problems with mine.

Chuanmei:               
"1: check whether whether YFP protein are there using YFP antibody. If
you don't have YFP antibody, try to use PCR and RT-PCR to check the
transgene insertion and transcription;
2: Use mutiple YFP, may be it's just the signal is too weak."

Rosemary White:
"Can you see the fusion protein on a Western blot or even just a dot blot?
This will tell you if you're getting any expression.  If no expression, then
you need to re-think your construct."

Jixiang Kong:
"Did you analyze the structure of your protein?? Sometimes it happens 
that GFP or YFP affects your protein structure either you use N-terminus 
or C-terminus. Consequently, they affect the expression of GFP or YFP."

Sam:
"I've been having the same problem exactly as you. Faith in any
 fluorescence detection 
actually happened in several proteins before. Changing different 
vector carrying the same YFP sometimes 'may' work while in my case not.
 Personally I feel that visibility of fluorescence protein depends on certain 
characteristic of our target protein itself. First, higher molecular weight 
protein is more difficult in expressing fusion protein than lower one. 
Second, the expression of our target protein might be very toxic to the 
cell resulting in the severe cell death after transformation. "

Marcus Heisler:            
"Sometimes these fusions just do not fold properly which ever end you  
fuse to GFP."
  
Yin, Ruohe:
"I have no much experience with that. I am just wondering if your
 Arabidopsis plants already contain any homologue sequence with 
your transgene, for instance 35S promoter could be in some T-DNA 
inserted lines, this sequence similarity may lead to silencing of your
 transgene. "


Andreas_Nebenf:
"You suspect that your protein may be unstable. If that's the case,
 then it is probably ubiquitinated and targeted for the proteasome.
 In this case, no YFP fluorescence would stay behind since the entire
 polypeptide chain would be degraded. You could check for this
 possibility by using a proteasome inhibitor (e.g., MG132).
 One thing to look for is whether there are any known targeting signals 
at the N or C terminus. If the YFP fusion blocks one of them, the
 protein would not reach its correct destination and probably be degraded.

We have also observed that some constructs lead to very low fluorescence 
levels while others seem more stable. In one case, we found a partial 
fusion to me more stable than the full-length fusion. Maybe the protein
 sequence can give you an indication of which regions to try. The partial 
fusion may not give you the "true" localization of the full-length protein, 
but you may learn something interesting about your protein.
Finally, sometimes transient expression can give signals that stable 
transformation does not tolerate."
   
Ignacio Rubio:                                                 
"If you do not see any YFP might be because of silencing of your transgene.
 It is very common than when overexpressing under 35S this happens.
 A good indication of that even is that plants are showing the phenotype 
of the KO of the linked gene to YFP. I would suggest you to change to 
another promoter or score more lines for YFP. I hope this could help you out."

Marcus:
"I would try an internal fusion site next. Try to find a region between 
functional domains."
        
Rosemary White:
"If your protein is unstable and rapidly degraded, the fusion protein will
suffer the same fate.  For example, SLN1-GFP is stable in aleurone nuclei of
dormant barley seeds, but is rapidly degraded - all gone within 10 min,
after application of GA.
See http://www.plantphysiol.org/cgi/content/full/129/1/191"












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