Abstract
The presented data provides additional information about the assessment of affinity purified nicotinic acetylcholine receptor (nAChR) rich membrane solubilized with long chain (16 saturated carbons) lysophospholipid with glycerol headgroup (LFG-16). The assessment of stability and functionality of solubilized membrane protein is a critical step prior to further crystallization trails. One of the key factors for this task is the appropriate choice of a detergent that can support nAChR activity and stability comparable to the crude membranes. The stability of the nAChR-LFG-16 complex incorporated into lipid cubic phase (LCP) was monitored for a period of 30 days by means of fluorescence recovery after photobleaching (FRAP) and the functionality was evaluated after its incorporation into Xenopus oocyte by means of the two electrode voltage clamp technique.
Highlights
Functionality and stability data of detergent purified nicotinic acetylcholine receptor (nAChR) from Torpedo using lipidic matrixes and macroscopic electrophysiology
The presented data provides additional information about the assessment of affinity purified nicotinic acetylcholine receptor rich membrane solubilized with long chain (16 saturated carbons) lysophospholipid with glycerol headgroup (LFG-16)
The stability of the nAChR-LFG-16 complex incorporated into lipid cubic phase (LCP) was monitored for a period of 30 days by means of fluorescence recovery after photobleaching (FRAP) and the functionality was evaluated after its incorporation into Xenopus oocyte by means of the two electrode voltage clamp technique
Summary
All steps were carried out in the cold room or on ice. In order to solubilize the crude membranes, these were thawed and mixed with a 10% (w/v) detergent solution and DB-1X Buffer (100 mM NaCl, 10 mM MOPS, 0.1 mM EDTA, 0.02% NaN3) for a final concentration of detergent 1–4%. The DB-1X buffer was added first, followed by the detergent and the crude membranes, which were added drop by drop. This solution was shaken slowly for 1 h and centrifuged at for 1 h at 40krpm and 4 °C. 12 mL of previously prepared bromoacetylcholine affinity resin (Bio-Rad Laboratories, Hercules, CA) in a 1.5 Â 15 cm Econocolumn (Bio-Rad Laboratories, Hercules, CA) was drained of storage buffer (40% Sucrose, 2 mM PMSF) was conditioned with 50 mL of ddH2O and 50 mL of 1.5 CMC detergent buffer before the supernatant prepared previously was added to the column. Our sample was eluted with 5 mL of 1.5 CMC detergent buffer and concentrated to 250 μL. Protein concentration was determined using a BCA Protein Concentration Assay (Pierce biotechnology, Rockford, IL) and a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was run to verify receptor purity
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