I'm looking to investigate if my protein (IRF3) is in monomeric or dimeric form. In order to effect some of its functions, I know that IRF3 must be phosphorylated and form a homodimer. So, I want to know how I can find out whether my samples contain the activated IRF3 in dimer form.
Hi Paula, We have described in details the protocols for IRF3 phosphorylation and dimerization detection in a recent paper in JoVE 2016. Let me know if you need more help. This is very standard procedure and works very well. Please contact me by email if you need: [email protected]
Article A High Resolution Method to Monitor Phosphorylation-dependen...
There are a number of ways to do this. Is this an in-vivo or in-vitro experiment? There are more possibilities in-vitro. If in-vivo, can make fusion proteins on IRF3?
Chemical crosslinking is a method that can be applied with purified proteins or with cells or subcellular material.
Dynamic light scattering (DLS) will tell you the oligomeric state of the protein in solution. It is a non-invasive technique that requires only little sample. As an alternative you might try size exclusion chromatography to give the apparent size.
Thanks everyone. @Nicole, they are in-vitro experiments. I read in a paper recently that separated the proteins by acrylamide gel electrophoresis under non-denaturing conditions and IRF-3 dimer formation was detected by immunoblotting. But, I'm curious as to how this might differ from Western Blot (if at all). I had been trying to check for IRF3 phosphorylation by WB, but it's been proving quite difficult and so, I'm hoping a dimerisation assay such as this might give me a faster result.
The most used methods today are Bret ( or fret) in transfected cella and proximilty ligation assays (pla) in fixed natural cells or tissue sections. For pla you may look movie in youtube dearching for olink and pla
Good luck
Western blotting normally begins with SDS-PAGE, which is denaturing and therefore is not useful for determining whether the protein is dimeric (unless you use a crosslinker).
Hi Paula, We have described in details the protocols for IRF3 phosphorylation and dimerization detection in a recent paper in JoVE 2016. Let me know if you need more help. This is very standard procedure and works very well. Please contact me by email if you need: [email protected]
Article A High Resolution Method to Monitor Phosphorylation-dependen...
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