In article <32EFC126.1CF4 at mailserv.cuhk.edu.hk>, Simon Chan <Simon> wrote:
>I am looking for methods where I can run a SDS-PAGE of a solubilized
>receptor preparation and then incubate the gel with the radioactive
>ligand, followed by autoradiography to reveal the receptor band. Can
>anyone point to me some good references or cookbook containing the
>protocols? Thank you.
I don't think that you're going to find many positive responses to this
question (correct me if you're wrong, of course). With regard to
7-transmembrane domain receptors, most will lose their ability to bind
ligand due the solubilization process and the electrophoresis.
Peturbation of the phospholipid environment in which the receptor
functions, denaturation, etc. are real problems.
Now, if you were to solubilize with a detergent that didn't munge up the
lipid environment of the receptor (we use CHAPS to solubilize the
thromboxane A2 receptor), and if you were to run non-denaturing SDS/PAGE
gels (protocols available in your favorite SDS/PAGE or protein cookbook),
you will increase your odds of success.
Incubation of the gel with the radioligand will depend on diffusion of the
ligand into the gel, it's ability to bind specifically, and your ability
to wash out all non-specific binding activity to reduce background. My
gut instinct tells me that this will be the most difficult part, because
when you try to wash out the unbound ligand, you will probably wash out
a great deal of the bound (unless the Kd is so low that the time constant
of binding is really long). I suppose that this would take some serious
experiments to find the right conditions.
Good luck,
-- Sam
--
Samuel C. Blackman ! InterNet : blackman at tigger.uic.edu
MD/PhD Student (4/8) ! Disclaimer: I speak for me, not UIC!
Univ. of Ill. at Chicago ! Quote : "Quandro potro io finir di stupire?"
Dept. of Pharmacology ! Phone : 312/996-4983 (lab) Fax: 312/996-1225
