hi, anyone please suggest me technique to detect presence of homodimer formation.
I need to know homodimer formation of mouse nfkb1 which has mol wt of 50 Kda and when i am infecting raw cells with H.pylori i need to check formation of p50 homodimer which has mol wt of 100 kda, the problem i am facing is my ab already gives two bands-one of p50 and another of p105(p50-precursor), so it will be difficult to differentiate between p100 and p105.
Could anyone has experience of detecting difference of minute 5 kda with any gel type.
please help
If your monomer precursor does not dimerize, and your mAb recognizes both native monomer and native dimer, you can switch to a SE-HPLC assay. Incubate your protein solution with saturating amounts of mAb (slight excess, ideally you should not have any free mAb or free protein). Monomer = 50+150=200 kDa; monomer precursor 105; homodimer = (50+150)*2=400. Make sure to use a column appropriate for your Mw range.
If mAb does not bind to precursor protein, you can get cleaner results by fluorescently label your mAb (pick your preferred label) and detecting fluorescence instead of A280/A214 (even better, both), this way you will discriminate fluorescent mAb bound to 50 kDa protein (and its dimer) and the 105 kDa precursor.
Good luck!
Thanks, but I am using polyclonal ab for p50 and how come monomer dimer will be 200, it should be 100?
Also, relative intensities of bands in a native vs denaturing PAGE may be informative.
Other possibbility is native IEF gel or isoelctrofocusing in capillary electrophoresis (best option in my opinion).
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