I am looking for an assay design that can help me analyse DNA (oligonucleotides) - protein interactions and provide some means to quantify these interactions.
Any help or direction will be much appreciated.
My personal favorite method for this purpose is fluorescence polarization. The oligonucleotide is synthesized with a fluorescent dye, such as FAM or TAMRA at one end. When the oligo binds to the protein, there is often an increase in fluorescence polarization due to the decreased mobility of the fluorescent dye. By titrating the protein concentration, you can measure the affinity of the interaction in solution. This technique requires either a spectrofluorimeter or fluorescence plate reader that is capable of making fluorescence polarization measurements.
Another widely used method is the gel mobility shift assay. Once again, the oligo is labeled with a fluorescent dye. The oligo migrates more slowly through the gel when bound to the protein than when unbound.
Another method is the thermal shift assay, which measures the change (usually increase) in the melting temperature of the protein as a result of binding to the oligo. This technique often makes use of an RT-PCR instrument.
There are many other techniques.
Here is a resource about this subject.
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/methods-detecting-protein-dna-interactions.html
@Adam B Shapiro thank you for your suggestions. I was initially thinking about EMSAs as well but wanted to explore other methods and see which one would work best. I'm glad I did so as I would not have thought about fluorescence polarization which seems like a pretty cool method. I was wondering if it would be able to give me insights regarding the kind of protein binding (monomer/ dimer, etc.)
EMSA would be a reliable method though you can also use SPR such as in the Biacore system.
I think that in principle it is possible to estimate the molecular weight of the complex by fluorescence polarization, but I don't think it is the best method. The best method in terms of accuracy, simplicity, and small sample requirement is probably size exclusion chromatography with multiangle light scattering (SEC-MALS). This requires specialized equipment, so you may have to look around to find someone who can do it.
Blue native polyacrylamide gel electrophoresis is a good method to try.
Another method that I like, because it only requires an SDS-PAGE setup, is chemical crosslinking using the water-soluble crosslinker BS3. By titrating the crosslinker concentration, you can get a pretty confident idea of the number of subunits in the complex.
Attached is a paper in which this method was used.
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays