Nature research paper: Autoregulation of GPCR signalling through the third intracellular loop
PMSF; 5 ml per confluent 15 cm dish) and incubated for 30 min on ice. Solutions were lysed gently in a chilled Dounce homogenizer . Nuclei and intact cells were separated from the lysate by centrifugation . Lysates were centrifuged . Spun-down lysate was resuspended in assay buffer . Pellets were then homogenized, first by passing through a 1 ml micropipette 10 times, then passing through a 26-gauge needle 10 times.
After Dounce homogenization, the following reagents were added to the membrane mixture : Cell membranes were prepared following the above procedure, but at a higher cell concentration , and without EGTA or DTT in the hypotonic buffer to prevent decreased efficiency of the click reaction. After Dounce homogenization, the following reagents were added to the membrane mixture : NaCl , KCl , MgCl, bovine serum albumin .
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