tre at wista.wistar.upenn.edu
Thu Jun 30 13:11:53 EST 1994
In article <199406301620.JAA04592 at cardinal.Stanford.EDU>,
richa at LELAND.STANFORD.EDU (Richa Behari) wrote:
> HI FOLKS,
> I am planning to do crosslinking studies with a membrane
> protein found in the malaria parasite within the red blood cell. The 23 kDa
> [deleted for brevity]
> If anybody out there has any protocols/ suggestions they would be very welcome.
Sounds like you want a photoreactive crosslinker so you can both
control the timing and can work under physiological conditions.
Imidoesters, NHS-esters and carbodiimides work best under more extreme
conditions than you want to expose your cells to, though it's
proven possible in my experience to hold cells in culture at pH 8-8.5
long enough to get an NHS-ester to work well. One can also control
the timing of the reaction easily with photoreactives--just keep
everything in the dark until you're ready to crosslink, then stick
it under a UV light or a camera flash. I've gotten pretty good
results with photoreactive aryl azides, which also don't require
any particular functional groups on your proteins--they supposedly
react with just about anything that's nearby. For what you're doing,
you alsoseem to want membrane permeability, and I don't know if
any of the photoreactive crosslinkers will cross the plasma membrane.
Talk to the people at Pierce Chemical in Rockford, IL. They make
all sorts of crosslinking goodies and can probably fit your needs pretty
exactly. Their tech service number is (800) 874-3723, and press 2 when
the recording answers.
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