Does anyone have reference material on GTP-Shift Assays? I have recently completed one and my results are not typical.
I was wondering if there are there any reference materials to help interpret the results?
Most central nervous system D2 dopamine receptors are coupled to their effectors by Go
Meisheng Jiang, Karsten Spicher, Guylain Boulay, Ying Wang, and Lutz Birnbaumer
Proc Natl Acad Sci U S A. 2001 March 13; 98(6): 3577–3582.
We reported previously that Go-deficient mice develop severe neurological defects that include hyperalgesia, a generalized tremor, lack of coordination, and a turning syndrome somewhat reminiscent of unilateral lesions of the dopaminergic nigro-striatal pathway. By using frozen coronal sections of serially sectioned brains of normal and Go-deficient mice, we studied the ability of several G protein coupled receptors to promote binding of GTP?S to G proteins and the ability of GTP to promote a shift in the affinity of D2 dopamine receptor for its physiologic agonist dopamine. We found a generalized, but not abolished reduction in agonist-stimulated binding of GTP?S to frozen brain sections, with no significant left–right differences. Unexpectedly, the ability of GTP to regulate the binding affinity of dopamine to D2 receptors (as seen in in situ [35S]sulpiride displacement curves) that was robust in control mice, was absent in Go-deficient mice. The data suggest that most of the effects of the Gi/Go-coupled D2 receptors in the central nervous system are mediated by Go instead of Gi1, Gi2, or Gi3. In agreement with this, the effect of GTP on dopamine binding to D2 receptors in double Gi1 plus Gi2- and Gi1 plus Gi3-deficient mice was essentially unaffected.