I have the suspect that a transcription factor interacts as a dimer with the DNA and my wish is to characterize it.
I'm wondering if it was possible to extract the protein nuclear fraction of cells and if any protein-protein complexes still exist in that fraction after the extraction is performed.
I'd like to use the NE-PER Nuclear and Cytoplasmic Extraction Reagents of the Pierce to extract the nuclear proteins: does any one have any experience concerning protein protein complexes in such a nuclear fraction extracted by mean of this kit?
After the extraction I'd like to perform an EMSA assay with that proteins and a specific DNA probe. Do the protein-protein-DNA complexes still exist during the EMSA run?
thank u Jason, so with this kinds of kits and assays the protein homodimers or heterodimers still exist?
Is your transcription factor supposed to bind the DNA specifically (sequence specific, for example)? You could employ MicroScale Thermophoresis: you use labeled DNA at constant concentrations and then you can just add a dilution series of your nuclear extract without any further purification. You could do the measurement even in cell lysate. With this assay, you could investigate protein binding to the DNA. If you are using the purified system, you could use very low protein concentrations and see dimerization on the DNA as soon as you add higher protein concentrations.
If you are intested, please feel free to contact me ([email protected]).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology