coimmunoprecipitation problems - (Jun/15/2006 )

Hello,

I'm doing coimmunoprecipitation and can anyone tell me if the protein beads (G) will be pulled down as also during the gel running, as I probably sucked out some beads together with the antigen into wells..Also, any tips for IP? Thanks in advance.

Marco

But the sample buffer is composed of Tris HCl which is pH 6.8, so I suppose it would only be very slightly acidic. Is there any thing which is acidic in the sample buffer?

Also I wonder if the protein target I'm interested in is not separated completely from its associates during the boiling procedure. I boiled the sample at 100 degree for 3 mins in a heat block of which the wells are full of water. There were some bands in the upper of the membrane, and that's why I suspect the proteins are not completely separated, so the protein I'm interested in was still in complex making it too large to be separated by stacking gel which is 4.5%.

Marco

You may try to elute the protein of interest from the protein G bead with 10mM glycine/HCl pH3.0, then neutralize it with 1M Tris.

I hope this may help.

Unfortunately, yes! They always come out of the beads and run into the gel if you use sample buffer to elute your protein

ha beads

They are no problem for running ur gel - simply spin down ur beads at 13000 rpm( they actually break at this speed), after u add elution buffer and incubate at 96C for 3min. Then use a long tip with small apperture and they should stay in the cup

Even if u have beads when u load, they get stuck in the well and the protein is eluted by the sds in the running buffer - remember its a SDS PAGE, n they are too large to really run in the gel

Elution - I guess u have some sds in ur elution buffer, thats sufficient or else add ß-mercaptoethanol, for sds page u can be as harsh as u want. I use lamelli buffer and cook for 3 mins that elute almost everything

praj