Here are 3 commonly used methods:

1. Transfect cells with a GFP construct of your protein and analyze the cells with confocal microscopy. Nuclei can be visualized by co-staining with TOTO-3 or DAPI (www.pnas.org/content/early/2010/01/19/0909086107)

2. Stain the cells after various stimuli and examine the nuclear translocation of your protein (www.pnas.org/content/109/44/18072.full)

3. Perform fractionation studies and study the presence of your protein in various fractions as a function of time (pubs.rsc.org/en/content/articlelanding/2012/IB/C2IB20114C; https://www.researchgate.net/publication/15668477_Biotherapy_of_B-cell_precursor_leukemia_by_targeting_genistein_to_CD19-associated_tyrosine_kinases)

Best of luck

Article Biotherapy of B-Cell precursor leukemia by targeting geniste...

Here are 3 commonly used methods:

1. Transfect cells with a GFP construct of your protein and analyze the cells with confocal microscopy. Nuclei can be visualized by co-staining with TOTO-3 or DAPI (www.pnas.org/content/early/2010/01/19/0909086107)

2. Stain the cells after various stimuli and examine the nuclear translocation of your protein (www.pnas.org/content/109/44/18072.full)

3. Perform fractionation studies and study the presence of your protein in various fractions as a function of time (pubs.rsc.org/en/content/articlelanding/2012/IB/C2IB20114C; https://www.researchgate.net/publication/15668477_Biotherapy_of_B-cell_precursor_leukemia_by_targeting_genistein_to_CD19-associated_tyrosine_kinases)

Best of luck

Article Biotherapy of B-Cell precursor leukemia by targeting geniste...

Hello Mr. Vasquez

Nice question. According to me, the immunocytochemistry not the only one method to analize migration of a protein. There are any other method, like immunofluorescens. I think immunofluorescens have a better result rather than immunocytochemistry alone. You can analize that migration using fluorescens microscopy with very clear image directly from the cell. That's all I know about that. Thank you.

Best regard - soni

fractionation and checking by western blotting may also be a method..

Like the others, immunocytochemistry is the way (seeing is believing!!), cellular subfractionation is a good complement but it could be really tricky. You'll have to come with very specific markers to convince people of your protein location!

Good luck!

Hi Fatih, thanks for your answer! However, if I want to analyze the migration of a drug to the nucleus, is it the isotopic radiation the best and/or the only approach I would have?

Dear,

As proposed by others, nuclear and cytoplasmic preparations are widely used in order to demonstrated the accumulation of a protein in the nucleus, followed by western blotting procedure. We use lamin A/C and GAPDH as marker of the nuclear and cytoplasmic homogenate to control cell fractionation. We started with 80 to 90 % confluent cells in 90mm-petri dishes . Cells are cultivated over 4 days before cell exposure to the drug for specified time. Ask me more wether You are interest by the protocol

Best Regards

Hi Andres ... I completely agree with the first answer by Fatih Uckun !! Have a good work!

as outlined by the colleagues immunhistochemistry and nuclear and cytoplasmic fractions are probably the first choice. Fractionating is most suitable if you work with cells. If you work on tissues samples, this is more challenging as more artefacts may occur during preparation.

In case you use tissue samples /tissue blocks for immunhistchemistry you may want to be aware that during fixation the localization of cell content (including proteins) may be altered by the diffusion of the fixative (not only lost, but altered!). This can result in nice artefacts of subcellular localization that look impressive but have nothing to do with the in vivo localization but merely reflect the effects of the diffusion of the fixation solution.

Radiolabelled drug: electron microscopy as suggested by Andras is great - but I would not hesitate to start with autoradiography and light microscopy - for 3H the subcellular localization is good enough to see if you drug is in the nucleus or in the cytoplasma.

Andres,

that is a little trickier. You need to label your drug but then it is possible that the labeling itself may alter the intracellular trafficking. After the drug is labeled, you can study the radiolabel accumulation in different cell fractions.

Article Biotherapy of B-Cell precursor leukemia by targeting geniste...

The answer depends on what facility you have to perform the experiment. All the above suggestions are correct and can be performed to compliment to see if the experiment has worked or not. Has anyone thought about using fluorescence recovery after photobleaching (FRAP)? You can find more info about the method and various publications at: http://www.microscopyu.com/references/frap.html

This is good for live cells if you are using one. Advantages of this method is, you can see the trafficking live under the microscope and also record it. Good luck.

I would go for endogenous tagging of the gene at question, N- or C-ter tagging depending on what affects less protein's function. This way you can study your protein's localization at levels of expression that are physiologically relevant. If you use a plasmid to express your tagged protein, you will overexpress it, and you step away of physiological conditions (nucleus transport proteins will not be overexpressed). Endogenous gene tagging is now a common and easy route. Please see the latest Nature Methods tech features on gene editing using sequence specific nucleases.

In case of proteins, it depends on what is your priority and what is the question. If you are interested in looking at the protein in an in vivo context, indirect immunoflourescence followed by confocal microscopy is the best and perhaps the only reliable option available. The disadvantage here is the method is snapshot static analysis.

If your interest is in studying the trafficking process and want to look at the dynamics of the nculeo-cytolpalsmic shuttle i.e. time or effect of drugs etc then make a flourescence tagged fusion protein overexpress it (best is to knock in) and do a live cell imaging.

Since you are interested in the drug (small molecule) and I presume florescent labeling is difficult and might affect the drug properties, radio-labeling is a better option. Following it you can do nuclear separation from cell lysates and count the radioactivity. Though being a cell biologist I rarely buy that kind of data. So I would do electron microscopy. I believe in what i see with my own eyes !! :p

I would suggest from my experience fractionation (nuclear/cytoplasm/mitochondria) but you have to be very careful about purity of fraction (use markers, but even some markers are not so specific for certain fraction, mostly for nuclear), another method is Amnis flow cytometry, which is stained for live cells for specific antibody and you can see nuclear localization.

Good luck and if you need more information about Amnis Imagestream system, I'll provide