On Thu, 30 Jan 1997 15:21:48 GMT, scb1 at midway.uchicago.edu (Samuel C.
Blackman) wrote:
>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.
>I would rather blot the receptors onto a membrane and do the
incubation with the ligand on the membrane-inthat way you will have
fixed the receptor and are not going to loose it during the
incubation. Ihtink thatm there there is almost ni chance to succeed in
triying to do the ligand binding with the gel!
>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
