Immunoprecipitation - problem with sepharose beads - (Feb/16/2007 )

Hi,

I got protein A sepharose CL-4B from amersham in a solid form. I swelled it in 1X PBS.

I added 30ul of protein A sepharose to pull the Antigen-Ab complex. When I added sample buffer and boiled at 95 deg C, the protein A sepharose solidified into a hard gel, that I had no supernatant to pipette out .

Please suggest what could be the problem.

Later, without doing IP, I just took the protein A sepharose beads, added sample buffer to it and boiled it again for 5 min. I observed the same result with the beads turning into a hard solid and there was no supernantant.

thanks.

Hi,

I got protein A sepharose CL-4B from amersham in a solid form. I swelled it in 1X PBS.

I added 30ul of protein A sepharose to pull the Antigen-Ab complex. When I added sample buffer and boiled at 95 deg C, the protein A sepharose solidified into a hard gel, that I had no supernatant to pipette out .

Please suggest what could be the problem.

Later, without doing IP, I just took the protein A sepharose beads, added sample buffer to it and boiled it again for 5 min. I observed the same result with the beads turning into a hard solid and there was no supernantant.

thanks.

this is really strange. It should not melt.
maybe you should write to amersham to ask them what's going on.
I use beads from amersham also, the fast flow, and I have no problem by heating at 95°C in laemmli buffer for 5 min.

the beads may not have been fully swollen when you added them to your solution. heating accelerates the swelling process.