Biology Reference
In-Depth Information
CHAPTER
Lipid-Binding Assays
11
David A. Gross
*
,{
and David L. Silver
{
Purification of Integral
Membrane Proteins and
*
Biochemistry Department, Albert Einstein College of Medicine, Bronx, New York, USA
{
Program in Cardiovascular & Metabolic Disorders, Duke-NUS Graduate Medical School
Singapore, Singapore
CHAPTER OUTLINE
Introduction and Rationale....................................................................................... 192
11.1 Materials........................................................................................................195
11.1.1 Buffers ...................................................................................... 195
11.1.2 Other Materials .......................................................................... 195
11.2 Methods .........................................................................................................195
11.2.1 Purification of Integral Membrane Proteins ................................... 195
11.2.1.1 Cell Culture of HEK293 Cells.............................................. 195
11.2.1.2 Cell Culture of Insect Cells ................................................. 196
11.2.1.3 Baculovirus Generation and Amplification........................... 196
11.2.1.4 Bacmid Purification and Storage ........................................ 198
11.2.1.5 Purification of Recombinant Proteins Expressed in
Insect Cells ..................................................................................... 199
11.2.1.6 Micellar Detergent Exchange Performed on Recombinant
Purified Proteins ............................................................................. 200
11.2.1.7 Biophysical Characterization of Purified Proteins................. 201
11.2.2 In Vitro Lipid-Binding Assays with Purified Recombinant Membrane
Proteins..................................................................................... 203
11.2.2.1 Binding Assays between Purified Recombinant FIT2
Produced in Insect Cells and TAG in Detergent Micelles................... 203
11.2.2.2 Binding Assays Between Purified FIT2 Overexpressed in
HEK293 Cells and Neutral Lipids in Detergent Micelles ..................... 205
11.2.3 Generation of Proteoliposomes..................................................... 206
11.2.3.1 Preparation of Proteoliposomes .......................................... 207
11.2.3.2 Limited Trypsin Digestion of Proteoliposomes ..................... 207
Conclusion and Applications.................................................................................... 208
Acknowledgment..................................................................................................... 209
References ............................................................................................................. 209
