I am interesting can anyone suggest how to track protein transport into the cells not using GFP or any big markers? I would like to minimize the effect of marker (keep size and function of protein of interest as possible to native), are some fluorescent markers which are small or what options should be used, except label with GFP kind markers?
A classic example to study protein import is to use pulse-chase analysis. One common method is to introduce radioactive amino acids that are incorporated into proteins and determine whether they are transported into the cell.
If you are looking for a marker smaller than GFP then HA is a common epitope tag that is only 9 amino acids. There are many vectors available that you can insert your gene of interest into that would tag your gene with HA.
Here is a suggestion. Hope it helps.
You could have a GFP molecule attached to the protein of your interest and optimize all the conditions to ensure that the protein goes inside the cell. Once the experimental conditions are optimized then you could do your expts with the protein without GFP attached to it hoping that things don't change very much when GFP is not tagged.
How do you want to investigate the transport? With life cell imaging or on fixed samples or with biochemical methods? This was not clear from your question. Could you please specify?
Cheers
Tilo
Hi Tilo,
I would like to use live cell imaging and fluorescent microscopy on fixed samples.
Thanks
David
Hi David!
I performed a lot of live cell imaging, but all intracellularly. For this purpose I tagged proteins with GFP and RFP and other fluorescent tags and eventually it always worked fine. You just have to perform a thorough screening first. In our projects we always tagged proteins C- and N-terminally and compared how they performed compared to each other and the endogenous protein (with antibody staining). You can also perform a test for enzymatic activity if your protein to make sure a FP tag does not influence this. As you say, a large tag can interfere with membrane transport, but with this I have no experience, unfortunately. There is also a technique where you only fuse a small part of GFP to your protein of interest and transfect the recipient cells with the other part. When they get in contact, the GFP assembles automatically and you get a fluorescence signal you can analyze.
There are other methods for labeling of proteins, there you need a certain amount of pure protein of interest which you can label with chemical dyes. They are way smaler than GFP, but I am not sure if their chemical properties have an influence on protein uptake in the cell. You can find a selection here: https://www.thermofisher.com/no/en/home/life-science/cell-analysis/labeling-chemistry.html#protein
For staining of fixed samples the golden standard is antibodystaining. You can also consider crosslinking of the protein of interest and use immunoprecipitation to analyze interaction partners.
I wish you the best of luck! Let me know if you wish to discuss this any further. Cheers, Tilo
As rightly pointed out tagging with GFP works and there are many examples where it was found to be successful. Alternatively you could tag one of the side chains of the amino acids in a protein (e.g. Lys, Cys etc) with a small fluorescent dye and image your samples. The dye will help you in imaging and tracking the protein inside the cells.
After labelling the protein, you could check its activity to ensure that tagging is not inhibiting its function or activity.
firstly, we could introduce radioactive amino acids, secondly， we could used link the a small fluorescent dye to the protein, for example FITC， Rhodamine B and so on. but the limtation of these way is that the small Molecule will change the active of target protein. the way of detection the target protein including Stochastic Optical Reconstruction Microscopy（STORM），Stimulated Emission Depletion（ STED），and so on.
Best wishes
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