:fauss at ITSA.UCSF.EDU ("Donald J. Fauss") wrote:
: > I need help on concentrating secreted proteins from cultured primary cells.
: > I've tried 70% EtOH precipitation but that didn't seem to work. I'll try
: > AmSO4 precipitation next but I'm wondering if I should include say 0.1%
: > TX-100 to help with the more hydrophobic proteins (maybe just to keep them
: > from sticking to the wall of the centrifuge tube?). I'm collecting them in
: > plain DME so I know the high salt concentration might prove a problem.
: > Probably I could detect the proteins with silver staining but I'm thinking
: > I'd need to be able to detect them with Coomassie staining before I'd have
: > enough protein to hopefully isolate species of interest. I'd like to hear
: > from anybody who has been successful at this.
One option is to collect the supernatant, spin to get rid of cellular
debris, and load it into dialysis tubing. Secure at both ends and place
in a tray with Aquacide (Calbiochem cat# 17851) sprinkled liberally under
and over the tubing. Place in the cold room and wait....
Aquacide is basically PEG with a MW of 500,000. You can also use PEG
20,000 from Sigma if you're using tubing with an appropriately low cutoff.
When the volume has decreased to a reasonable level, wash off the now
gooey aquacide with water and dialyse your sample into an appropriate
buffer if you so desire.
I picked this technique up from someone who post-doc'd with Gail Martin at
UCSF in the early 80s so you might be able to wander down the hall if you
need assistance. The technique is pretty straight forward though.