I am trying to find a method to observe RNA binding for an alkaline protein (pI 9.2), but if I run the protein-RNA complex in a conventional native-TBE PAGE(4-6%), I find the protein stuck onto the well, thus I am unable to see a conclusive shift in the RNA migration. Can you suggest any other buffering systems or binding assays that can be used to solve this problem?
Hi. You can try tris-glicine gels or if your RNA-protein complexes are quite big agarose gels.
A general problem with EMSAs is that they quite often indicate higher affinities, this is mainly based on the so-called caging effect caused by the gel matrix. MicroScale Thermophoresis (MST) in contrast is a technology to investigate biomolecular interactions free in solution without any immobilization or matrix present. In addition, affinity measurements can be performed in any buffer.
This means you are independent of the pI of your protein!
For more information on the technique, you can watch a 3-minutes movie
http://www.nanotemper-technologies.com/news-events/news/article/watch-a-3-minutes-movie-to-explore-microscale-thermophoresis-mst/
or contact me directly via [email protected]!
Arwa A. Abugable I cannot remember to have much success with any of the methods...I did try the agarose by having the combs in the middle of the gel rather than the extremity... I did see some differences there....but did not pursue much after that..
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