labtek microscope chamber  (Labtek)

Journal: Scientific Reports

Article Title: The lateral distance between a proton pump and ATP synthase determines the ATP-synthesis rate

doi: 10.1038/s41598-017-02836-4

Figure Lengend Snippet: Confocal scanning microscope images of a GUV in which two fluorophore-labeled proteins were reconstituted. ( A ) Detection of cyt. bo 3 labeled with ATTO 647N. The focal plane is at the middle of the vesicle. ( B ) Detection of ATP-synthase labeled with ATTO 594. ( C ) Combined image of ATTO 594 and ATTO 647N detection. ( D ) An image of the top of the vesicle, which is the focal plane used for the FCS measurements. The lipid composition of the vesicle was 99% DOPC and 1% DPPE functionalized with a biotinyl head group. The GUVs were immobilized on a streptavidin-coated cover slide and the solution around the vesicle was 10 mM HEPES pH 7.4, supplemented with 10 mM NaCl and 100 mM glucose.

Article Snippet: The two proteins, cyt. bo 3 and ATP synthase, were co-reconstituted into the GUVs using a mild detergent treatment with DDM (or Na-Cholate), using a protocol modified from ref. . First, the protein stock solutions with 1 mM DDM (or 46 mM Na-Cholate) were mixed with 20 μl GUV-solution to a final concentration of 0.05–0.25 μM protein and 0.05 mM DDM (2.3 mM Na-Cholate) and incubated at room temperature for 30 min. Then, the proteo-GUVs were diluted 20 times in a 10 mM HEPES buffer (pH 7.4) containing 100 mM glucose and 10 mM NaCl, transferred to a LabTek microscope chamber coated with streptavidin (to immobilize the GUVs by the interaction with DPPE-biotinyl) and further incubated for 2 h. The dilution gave a final detergent concentration of 2.5 μM DDM (0.12 mM Na-Cholate) in the sample.

Techniques: Microscopy, Labeling