Dear all,
I need your valuable suggestion in the following.
I am tracking a viral protein in an infected cell. My model cell line is Jurkat T cells which are CD4+ suspension cells. Now, this viral gene has been stably chromatinized at a single copy per the host cell genome. I am following the subcellular localization of the protein at multiple time points by confocal. Indirect immunofluorescence was performed at different time points with an Alexa 568 tagged secondary antibody. I also find that the protein is present at the PM, cytosol and nucleus at diff time points and the result is reproducible. But I am concerned about what the reviewers might ask additionally as a supporting data.
Can you think of any loopholes in the experimental design and what additional experiments can be done to prove my point?
Its possible that viral proteins present at different cellular compartments at different stages of their life cycles. I would first suggest known literature about the proteins and the virus. If you have proper markers for your subcellular compartments, its a good control for your observation. However reproducibility of your results should be enough. For further confirmation you can carefully isolate the nucleus or cell membrane and do a western blotting. If you have a live cell imaging facility, you can attach your cells and follow a group of cells for several hours. I hope this helps you. Goodluck
Hello, Sutanuka.
They would raise questions;
1) From the beginning, you should justify why you are interested in the protein and its cellular distribution.
2) why you didn't do live tracing the viral protein-GFP in over-expressing cells, providing more reasonable evidences for time-course distribution of it (although it is not completely direct, yet) in live single cells, giving ER-, Golgi-biomakers or some other organelles.
3) If he protein have such extensive distributions within the cells, then you should show functional studies associated with its distribution whether it's an RTK or other oncoprotein as an independent protein product, demonstrating that it is not a fused gene product, by being inserted into the middle of or at the end of host genomic gene sequence.
4) By having the protein-GFP-overexpressed cells you could accomplish above functional study in live cells by treating various signals (cell activation via any receptor or inactivation via various inhibitors). This will define what is the function(s)of the protein or why they show up in all corners of the cell.
5) If the protein is an independent product, it can be visualized on Western blot by its molecular mass.
6) Is the protein interacting with other cellular protein? IP will help this answer.
7) Doesn't it have any signal peptide sequences or Does it have every signal peptide sequences in its intrapeptide sequence because it show PM, cytoplasm and nucleus distribution.
Just think about them. Best wishes.
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