GFP can not be observed! Help. - (Oct/19/2008 )
Hi all,
Thanks you for your advice on how to select the true transformant. Now I have got several T1 heterozygous GFP arabidopsis by PCR. But the new problem comes: 
I take a leave from the plant and mounted in water and use fluorsecent microscope to observe the signal. But I did not found bright green siganl there. Insead, there is only some background green light at the edge of cell. This is also found in wt plants. I also checked in confocal microscope, the same.
Here is some information about my construct. I used pMDC43 binary vector, which gfp is fused to N terminal of my protein. My protein contain a tranmembrane domain. I sequenced the junction between gfp6 and my protein, it is in frame. I used Agrobacterium for transformation and floral dip method.
My question is:
what may be the reason? What should I do next?
Does any one also have such experience?
Thanks a lot.
P.S.
Many thanks to smu2 and frozen for your mentorship.
If the protein is transmembrane, and GFP is on the N-terminus, that means that GFP must act as a transmembrane signal. You know, all transmembrane proteins have few hydrophobic amino acids at their N-terminus which signal ribosomes that proteins must be synthesized on a membrane (for example ER membrane). If GFP is on N-terminus, it might block this signal sequence and your protein is not synthesized on membrane but elsewhere, thus, not be able to perform its normal function. Cell then degrades these aberrant proteins...
of course, this is just a hypothesis 
. M.
Thanks you for your advice on how to select the true transformant. Now I have got several T1 heterozygous GFP arabidopsis by PCR. But the new problem comes:
I take a leave from the plant and mounted in water and use fluorsecent microscope to observe the signal. But I did not found bright green siganl there. Insead, there is only some background green light at the edge of cell. This is also found in wt plants. I also checked in confocal microscope, the same. Here is some information about my construct. I used pMDC43 binary vector, which gfp is fused to N terminal of my protein. My protein contain a tranmembrane domain. I sequenced the junction between gfp6 and my protein, it is in frame. I used Agrobacterium for transformation and floral dip method.
My question is:
what may be the reason? What should I do next?
Does any one also have such experience?
Thanks a lot.
P.S.
Many thanks to smu2 and frozen for your mentorship.
I agree with Frozen. Perhaps GFP interfere when fused to the N-ter of your protein.you could do the transient expression before doing transforming to the plants.
Stone
Thanks a lot. Yeah, I think transient expression is a easier way since the in planta transformation take me half years.
I will try mesophyll protolast developed by Dr. Jen Sheen. But this techniques may need several months to a year to master. Will particle bombard quicker and easier than protoplast?
I 'v never done transient expression before.
Also, I read a paper talking about the degradation of vacule targeted GFP-fusion protein is accelerated under light.
Thanks you for your advice on how to select the true transformant. Now I have got several T1 heterozygous GFP arabidopsis by PCR. But the new problem comes:
I take a leave from the plant and mounted in water and use fluorsecent microscope to observe the signal. But I did not found bright green siganl there. Insead, there is only some background green light at the edge of cell. This is also found in wt plants. I also checked in confocal microscope, the same. Here is some information about my construct. I used pMDC43 binary vector, which gfp is fused to N terminal of my protein. My protein contain a tranmembrane domain. I sequenced the junction between gfp6 and my protein, it is in frame. I used Agrobacterium for transformation and floral dip method.
My question is:
what may be the reason? What should I do next?
Does any one also have such experience?
Thanks a lot.
P.S.
Many thanks to smu2 and frozen for your mentorship.
I agree with Frozen. Perhaps GFP interfere when fused to the N-ter of your protein.you could do the transient expression before doing transforming to the plants.
Stone
I will try mesophyll protolast developed by Dr. Jen Sheen. But this techniques may need several months to a year to master. Will particle bombard quicker and easier than protoplast?
I 'v never done transient expression before.
Also, I read a paper talking about the degradation of vacule targeted GFP-fusion protein is accelerated under light.
Thanks you for your advice on how to select the true transformant. Now I have got several T1 heterozygous GFP arabidopsis by PCR. But the new problem comes:
I take a leave from the plant and mounted in water and use fluorsecent microscope to observe the signal. But I did not found bright green siganl there. Insead, there is only some background green light at the edge of cell. This is also found in wt plants. I also checked in confocal microscope, the same. Here is some information about my construct. I used pMDC43 binary vector, which gfp is fused to N terminal of my protein. My protein contain a tranmembrane domain. I sequenced the junction between gfp6 and my protein, it is in frame. I used Agrobacterium for transformation and floral dip method.
My question is:
what may be the reason? What should I do next?
Does any one also have such experience?
Thanks a lot.
P.S.
Many thanks to smu2 and frozen for your mentorship.
I agree with Frozen. Perhaps GFP interfere when fused to the N-ter of your protein.you could do the transient expression before doing transforming to the plants.
Stone
Hello again
I think protoplast transformation is not very hard as you think. We often use PEG instead of particle bombard.
and you can also try doing transient expression in tabacco leaf. It is also a good method. It works also well in our lab.
Stone
Thanks you for your advice on how to select the true transformant. Now I have got several T1 heterozygous GFP arabidopsis by PCR. But the new problem comes:
I take a leave from the plant and mounted in water and use fluorsecent microscope to observe the signal. But I did not found bright green siganl there. Insead, there is only some background green light at the edge of cell. This is also found in wt plants. I also checked in confocal microscope, the same. Here is some information about my construct. I used pMDC43 binary vector, which gfp is fused to N terminal of my protein. My protein contain a tranmembrane domain. I sequenced the junction between gfp6 and my protein, it is in frame. I used Agrobacterium for transformation and floral dip method.
My question is:
what may be the reason? What should I do next?
Does any one also have such experience?
Thanks a lot.
P.S.
Many thanks to smu2 and frozen for your mentorship.
I wouldn't necessarily give up on these transformants until you check the T2 - also I would look at root tips rather than leaves because it's much easier to detect signal when you don't have chlorophyll interference. You could do a western on leaves if they aren't at the stage yet where you can get T2 seedlings and you should be able to detect the protein if it is indeed being translated properly.
Good luck,
smu
I agree, do western. M.