Working on various deletion/truncation/interface mutants of a membrane protein and trying to determine the effect of those on there oligomeric state. Is there any way to determine oligomeric state of a membrane protein (or soluble) within the cell and without protein purification? I have already thought about cross linking and split GFP. If any one of you have some other idea please share with me .
you can try FLASH fluorescence. This involves putting a bicysteine motif onto your protein, which upon dimerization and formation of a tetracysteine binds arsenic and becomes fluorescent. It can be done in vivo.
http://www.nature.com/nchembio/journal/v7/n9/full/nchembio.624.html
The proximity ligation assay may be another approach
http://www.nature.com/nmeth/journal/v3/n12/full/nmeth947.html
However, with both technques it may be difficult to tell the actual oligomerization state beyond monomer or oligomer (maybe through intensity analysis?).
You could try MicroScale Thermophoresis (MST)! MST is a technique based on the directed movement of molecules in temperature gradients. Since this movement depends on the size, charge and hydration shell of molecules, you can distinguish bound and unbound molecules and therefore you will be able to determine binding affinites.
For your question, you would need to express your protein as fluorescent fusion protein (such as GFP or mCherry). You do not have to purify the protein, but you can just use the cell lysate. Depending on how tight the oligomers form, the protein should be monomeric at low concentrations. Then you add increasing amounts of non-fluorescent protein, which again does not need to be purified. Upon oligomerization, you should then see a different thermophoresis signal for your fluorescent target protein. As a negative control I would recommend to employ cell lysate without overexpression of your target protein.
Please contact me via [email protected] for further questions or consult our website containing references and publications:
http://www.nanotemper-technologies.com/applications/
If you can label your proteins with a fluorescent protein, some kind of fluorescence fluctuation analysis such as Number & Brightness or RICS should be the most powerful tool to answer your question IN VIVO. The problem is that it requires quite sophisticated equipment. See e.g.
- Mapping the Number of Molecules and Brightness in the Laser Scanning Microscope. Michelle A. Digman et al. Biophysical Journal 2008, Volume 94, 2320-2332
- Determination of Membrane Protein Transporter Oligomerization in Native Tissue Using Spatial Fluorescence Intensity Fluctuation Analysis. Mikhail Sergeev et al. PLOS One 2012; DOI: 10.1371/journal.pone.0036215
- Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy. Yan Chen et al. PNAS 2003, 100 , 15492–15497
If everything else fails, do not forget good old Blue Native Electrophoresis.
I agree with Pierre's suggestion. The use of FFS methods to study protein oligomerization is pretty straightforward now. See also James et al. (2012) Biophys J.102:L41-3. Number and Brightness Analysis of LRRK Oligomerization in Live Cells. or Jameson, D.M., James, N.G. and Albanesi, J.P. (2013) Methods Enzymol. 519:87-113. Fluorescence Fluctuation Analysis Approaches to the Study of Receptors in Live Cells.
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