Biology Reference
In-Depth Information
CHAPTER
Receptors
24
David Salom
*
, Pius S. Padayatti
*
, and Krzysztof Palczewski
*
,{
*
Crystallization of G
Protein-Coupled
Polgenix Inc., Cleveland, Ohio, USA
{
Department of Pharmacology, School of Medicine, Case Western Reserve University,
Cleveland, Ohio, USA
CHAPTER OUTLINE
Introduction ............................................................................................................ 452
24.1 Crystallization of Bovine Rhodopsin .................................................................453
24.1.1 Materials ................................................................................... 453
24.1.1.1 Rod Outer Segment (ROS) Isolation with Sucrose Gradient ..453
24.1.1.2 Nonyl-glucoside/Zn(OAc)
2
Extraction of Rhodopsin..............454
24.1.1.3 Immunoaffinity Purification .................................................454
24.1.1.4 (NH
4
)
2
SO
4
-induced Phase Separation .................................454
24.1.1.5 Crystallization .....................................................................454
24.1.2 Methods .................................................................................... 455
24.1.2.1 ROS Isolation with Sucrose Gradient....................................455
24.1.2.2 Nonyl-glucoside/Zn(OAc)
2
Extraction of Rhodopsin..............455
24.1.2.3 Immunoaffinity Purification of Rhodopsin ............................455
24.1.2.4 (NH
4
)
2
SO
4
-induced Phase Separation .................................456
24.1.2.5 Crystallization .....................................................................458
24.2 Crystallization of
b
2-AR ..................................................................................460
24.2.1 Materials ................................................................................... 460
24.2.1.1 Solubilization of Membranes ...............................................460
24.2.1.2 Immunoaffinity and Gel Filtration Chromatography...............461
24.2.1.3 Crystallization .....................................................................461
24.2.2 Methods .................................................................................... 461
24.2.2.1 Protein Purification .............................................................461
24.2.2.2 Mesophase Crystallization ...................................................463
24.3 Discussion......................................................................................................465
Acknowledgments ................................................................................................... 465
References ............................................................................................................. 466
