Magnetic beads with FastChip? - Possible? Anyone tried? (Mar/18/2008 )
Is it possible to replace magnetic beads for the agarose beads in the FastChip protocol? Do you add the beads with the antibody as you do with the Upstate MagnaChip kit or would you do the chromatin/antibody incubation and then add the beads later as is normally done with agarose beads?
On a related question: the FastChip protocol uses a centrifugation step after the antibody incubation to clear the chromatin. I'm assuming this clears non-immune complexes that may precipitate in the chromatin/antibody mixture. Do you think this can be easily adapted to the MagnaChip kit? Will the MagnaChip kit work if you incubate the antibody/chromatin without the beads, spin, and then add the magnetic beads? How long would you incubate with the beads after the spin? I don't see why this wouldn't work, but the kit protocol is pretty clear about adding the beads and the antibody simultaneously.
I'm getting qualitatively similar results using the Upstate MagnaChip kit, but the enrichment of my transcription factor is all over the map, so it isn't quantitative. I'm hoping that adding in some sort of clearing step will help as I've read it is useful in the FastChip protocol. I'd also like to start using the FastChip protocol if it can be used with the magnetic beads (they are just so much cleaner than agarose).
Any suggestion on what brand of magnetic beads to use if trying them with the FastChip protocol?
Thanks!
Kurt
On a related question: the FastChip protocol uses a centrifugation step after the antibody incubation to clear the chromatin. I'm assuming this clears non-immune complexes that may precipitate in the chromatin/antibody mixture. Do you think this can be easily adapted to the MagnaChip kit? Will the MagnaChip kit work if you incubate the antibody/chromatin without the beads, spin, and then add the magnetic beads? How long would you incubate with the beads after the spin? I don't see why this wouldn't work, but the kit protocol is pretty clear about adding the beads and the antibody simultaneously.
I'm getting qualitatively similar results using the Upstate MagnaChip kit, but the enrichment of my transcription factor is all over the map, so it isn't quantitative. I'm hoping that adding in some sort of clearing step will help as I've read it is useful in the FastChip protocol. I'd also like to start using the FastChip protocol if it can be used with the magnetic beads (they are just so much cleaner than agarose).
Any suggestion on what brand of magnetic beads to use if trying them with the FastChip protocol?
Thanks!
Kurt
I actually had some Dynal magnetic beads to try with Fast ChIP but got never got a chance to do the experiment. The one thing I can say about these beads is that they have a much lower capacity than the Amersham Fast Flow agarose. This probably won't be a problem, since the amount of beads we use in Fast ChIP FAR exceeds the binding capacity required for the amount of antibody used. Still, you might want to calculate and make sure that the amount of beads you use will be sufficient (even saturating) for the amount of antibody you want to use.
As far as when to add the beads, I would add the beads after the antibody/chromatin incubation and centrifugation. If you add them before, you won't be able to get rid of the unspecific protein-DNA complexes which may precipitate during the ultrasonic bath incubation. In addition the ultrasonic bath might start to fracture the beads (I don't know, I've never subjected the beads to this). In any case, if you have any other questions about Fast ChIP, let me know.
Joel