These pictures should indicate the presence of the GFP and your protein

The quality of the image does not allow to zoom in and see it appropiately, but I would suggest ER localization. Have you used BFA? Alternatively (less probable) cytoplasmic localization.

Thanks everyone,

I can show you some bright field pictures as well here:

Ok, in case somebody follows this question later, I ran a complementary experiment, I used a nuclear marker to double check where that central signal is coming from and it merged with my green signal which means that dot is the nucleus. I'm also attaching one picture for you :)

Hi Shirin Mirlohi,

Yes, you have GFP in the nucleus. But look like you also have GFP in the cytosol (?). The GFP in the cytosol could be pushed by a huge vacuole to the side of cell membrane. So you can see the green circle around the cell membrane.

Except for nucleus marker, have you tried other organelle markers to test?

Hi Yuan,

Actually the cytosolic localization has been reported before many times so i'm not focusing on that. I'm only working on the nucleic localization for my purpose :)

So, your protein can localize to both nucleus and cytosol organelles?

Yes, exactly. 

@ Shirin,

Just wondering, does your gene have 'NLS' or nucleus-localization-signal peptide?

No it does not :) i'm running a new experiment now, added NES and NLS signals to this gene to study it's localization and whether or not these two different localizations lead to any special phenotype in my plants.

1. That was my thought (no NLS). Otherwise, the protein should all be targeted to nucleus.

2. When you add NES/NLS to your protein, which end of the protein you are going to add them?  Some research results indicated that it is more effective to add them at the C-terminus of a protein, while some demonstrated that it is more effective to add them at the N-terminus of a protein. Some researchers even add these signal peptides (ex. NLS) to both ends of a protein [ex. NLSsv40-protein-NLSsv40]. Some add two different NLS to both ends of a protein [ex.  NLSSV40-GFP-NLSEGL-13] (see attached paper). So, it will be safer if you can build different constructs and test them.

Hello :)

Oh thank you so much Yuan, I really appreciate your suggestions. Yeah that seems like a smart idea to try the tag at both ends. I usually try making both constructs  Protein-NLS/NES and NLS/NES-protein (i do the same for my BiFC experiments as well to double check the results), but I never thought of adding the tag to both ends, actually that seems even better. Thank you Yuan, I always enjoy reading your helpful comments.

@ Shirin,

NLS/NES at the two ends of a protein can be better for nucleus targeting, but no guarantee. That is why you need to test it and find out.

@ Shirin,

1. After you construct (1) NLS/NES-protein(x), (2) protein(x)-NLS/NES and (3) NLS/NES-protein(x)-NLS/NES, what kind of plant you are going to use for transforming these constructs? The wild-type or a mutant (knockout function of protein(x))? And why?

2. Is there any protein(x) knockout mutant line out there? Does knockout this protein(x) lethal to the plant?

**Protein(x) is the protein of your interest, which targets to both nucleus and cytosol

@Yuan,

1. I'm going to transform the K.O. plants which I already have (maybe it would be a good idea to transform the WT as well as a control)

2. no the K.O plants stay alive but show an obvious phenotype.

@ Shirin,

1. What will be the 'control'?

@yuan,

the control would be the non-transformed knock out version.

@ Shirin,

1. I did not see your last response until today. RG did not send me new update at all after you posted your answer.

2. By the way, I was surprised to see people engineer a protein with 3 NLS. Two NLS on one end and the other on the other end. I was doing some article reading about CRISPR/Cas9 system, and came across with this kit (see attachment)

See page 1, the Cas9 is engineered to have 3 NLS:  Alt-R™ S.p. Cas9 Nuclease 3NLS

Regarding the 'control":

Now I know. So, you meant to 'transform a WT as a control', not 'as well as a control' (your early post). If you say 'as well as', it seems that you are going to transform both 'WT' and 'a control' (2 transformations). I was a bit confused earlier.

So, you will have those lines to compare?:

1. WT, 2. Mutant, 3. WT transformed with your NLS-construct(s), 4. Mutant transformed with your NLS-construct(s), 5. WT transformed with your NES-construct(s), 6. Mutant transformed with your NES-construct(s). That is a lot work.  

Happy coming New Year!!

@yuan,

yes that's exactly what i'm trying to do :)

Happy new year to you too.

I think this protein localized all over the protoplast cells. I can see them in the plasma membrane, chloroplast, ER, and nuclus.