In article <37BA531E.E23E0F0B at home.com>, Neal Melvin <nrmelvin at home.com> wrote:
> After performing an IP, do you run the actual beads themselves through
> the gel?? Or is there a step that strips the protein A/G and bound
> antibody/antigen off the beads, and the resulting supernatant is run
> through the gel???
The Ab/Ag complex will dissociate from protein A/G agarose beads after you
add SDS-PAGE sample buffer. Incubate at RT for 15 min and spin for 1 min
at 6000 rpm. Load only the supernatant.
However, I suggest you load everything including the beads (with a long
thin micro-pippet) after 15 min incubation if the well volume is allowed,
as this reduces the loss of your protein. Rinse the tube with 10ul sample
buffer when necessary. The beads will remain in the well during
electrophoresis. Hope this helps.
Mao Dong
Dongm at mayo.edu
