Magnetic beads - (Jan/19/2006 )
I want to purified proteins whose interact with a tagged protein . I have magnetic beads on whose the antibody directed againts the tag is fixed. Then I incubate those beads with a protein extract.
I wanted to know if somebody have already try to do that, and if somebody have a protocol
thanks in advance
I've done something similar with his-tagged proteins and DynaBead Talon paramagnetic beads.
The beads have a cobalt attached to them, and thus bind the his-tag (as nickle does). This is somewhat analogous to your experiment, except you have an Ab instead of a cobalt, and a ligand instead of a his-tag...
It worked well, and wasn't hard. I'm at home and don't have the protocol in front of me, but it was essentally equilibrate (wash) the beads (50 ul) in 700 ul binding/wash buffer (50 mM Na phosphate buffer, pH 8.0, 500 mM NaCl, and some Tween-20 -- 0.01% perhaps?), resuspend the beads in 600 ul binding/wash buffer, then add your sample (100 ul) to the beads. Bind the sample by incubating (RT or 4C or 37C depending on your application) for 30 minutes while mixing (end-over-end). Pull the beads down with a magnet, pipette off the supernatant and wash (resuspend the beads in 700 ul binding/wash buffer, pull them back down with a magnet, decant the supe) four times.