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Proving calcium dependent protein interaction


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#1 Reverse Transcribed

Reverse Transcribed

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Posted 09 March 2010 - 04:40 AM

Hi all!

May be someone could direct me to the right path here..

I need to prove the calcium dependent interaction of several mammalian proteins. I have some FRET data, but need to verify it, using in vitro methods.

Having only limited background in protein biochemistry, my initial idea was to make a native gel electrophoresis. So I went ahead and generated plasmids for His-tagged purification of these proteins in E. coli. Initial bacterial induction and small scale purification worked nicely and I need to scale it up now. In the end I plan to mix the proteins in different buffers (with different Ca2+ concentration) and run on gel. Yet, I am starting to have doubts on whether this is the right method. Namely the proteins are likely to form homodimers, but may be also tetramers. So in case the gel runs nicely, I might end up with a large number of bands, making it difficult to interpret the results.

So my second thought now was to make a pull-down experiment. Bind the His-tagged protein to the beads and load tissue extract, where fluorescently labelled other protein is expressed. Finally I could elude it from the beads and detect the presence of the other protein, using an antibody against the fluorescent protein. The advantage of this method is that I might get at least one protein directly from the mammalian tissue, having the right folding state. Here the pitfall is that the fluorescently labelled protein might all bind to cell membranes, when I homogenize the tissue (because of Ca2+) and I might not get it out. I could probably add EDTA, but it will likely inhibit the interaction of the proteins on the Ni-NTA beads.

Any ideas, on which method to choose?
How to extract calcium sensitive proteins from tissue samples in native conformation? May be if I could somehow do it very fast and transfer to the beads?

Thanks a lot!

RT




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