Abstract
This experiment was a study of the different properties of a lipid bilayer. We used fluorescent microscopy to study the lateral diffusion of lipids and binding properties biotin and anti-biotin. The effect cholesterol has on the fluidity of the membrane was tested using Fluorescent Recovery After Photobleaching (FRAP). We used a Nikon Eclipse 50i upright fluorescent microscope with a Digital Sight DM-2MBW CCD camera to take images of the bilayer after photobleaching. Using these images we were able to determine the diffusion coefficients for the bilayer to be , without cholesterol and , with cholesterol. Using the same microscope and camera alongside an orange Helium-Neon Laser at 594 nm we studied the binding of biotin and anti-biotin …show more content…
To clean the wells, we used diluted 7X detergent, we rinsed them with high purity water and methanol, blotted them dry with lens paper. The wells were then assembled by sticking and removing a piece of scotch tape from each face of the well and then sticking the well to glass coverslips. While the wells were being cleaned and assembled the dilution team diluted the Texas Red labeled anti-biotin antibody into five different 70 µL solutions with concentrations of 8 µM, 4 µM, 2 µM, 1 µM, and 0.5 µM, and one 140 µL solution with a concentration of 0.25 µM. They did this by pipetting the Texas Red labeled anti-biotin into six different centrifuge tubes and then adding the necessary amount of phosphate buffered saline (PBS pH 7.4, 17mM NaCl, 17.5mM Na2HPO4, 1.83mM KH2PO4) to create the required concentrations. The cleaning team then prepared the bilayers in the PDMS wells while the dilution team prepared the microscope. To prepare the bilayers 30 µL of vesicle solution 3 (93.5 mol% POPC / 1.5 mol% PEG-DPPE / 5 mol% biotin- cap-DPPE vesicle solution) was put into 6 of the PDMS wells and 30 µL of vesicle solution 4 …show more content…
The microscope only needed to be focused the only light used for this experiment came from the microscope itself and not an external laser. The 4 PDMS wells were cleaned the same way as previously described. This time the glass coverslips was scratched with a diamond etcher before sticking the well to it. Two of the wells were then filled with 30 µL of vesicle solution 1 (contains: 99.5 mol% POPC with 0.5 mol% Texas Red labeled DHPE) and the other two with vesicle solution 2 (contains: 69.5 mol% POPC, 30 mol% cholesterol, and 0.5 mol% Texas Red labeled DHPE). These were then incubated for 30 minutes. After incubation the 30 µL of vesicle solution were suctioned out of the wells and the wells were refilled with 30 µL of PBS buffer. The wells were then sealed with another glass coverslip. The samples were flipped over before they were viewed so the bilayer would be on the top of the well. The microscope was focused and the sample was viewed with the 40x objective. We began photobleaching the samples at this point; they were photobleached with a green light at 100% intensity. We photobleached the sample without cholesterol for 3 minutes, we then took the light intensity down to 10% to take images of the sample and shuttered the light after each picture to prevent further photobleaching of the sample. For the first 10 minutes a photo was
This experiment was a study of the different properties of a lipid bilayer. We used fluorescent microscopy to study the lateral diffusion of lipids and binding properties biotin and anti-biotin. The effect cholesterol has on the fluidity of the membrane was tested using Fluorescent Recovery After Photobleaching (FRAP). We used a Nikon Eclipse 50i upright fluorescent microscope with a Digital Sight DM-2MBW CCD camera to take images of the bilayer after photobleaching. Using these images we were able to determine the diffusion coefficients for the bilayer to be , without cholesterol and , with cholesterol. Using the same microscope and camera alongside an orange Helium-Neon Laser at 594 nm we studied the binding of biotin and anti-biotin …show more content…
To clean the wells, we used diluted 7X detergent, we rinsed them with high purity water and methanol, blotted them dry with lens paper. The wells were then assembled by sticking and removing a piece of scotch tape from each face of the well and then sticking the well to glass coverslips. While the wells were being cleaned and assembled the dilution team diluted the Texas Red labeled anti-biotin antibody into five different 70 µL solutions with concentrations of 8 µM, 4 µM, 2 µM, 1 µM, and 0.5 µM, and one 140 µL solution with a concentration of 0.25 µM. They did this by pipetting the Texas Red labeled anti-biotin into six different centrifuge tubes and then adding the necessary amount of phosphate buffered saline (PBS pH 7.4, 17mM NaCl, 17.5mM Na2HPO4, 1.83mM KH2PO4) to create the required concentrations. The cleaning team then prepared the bilayers in the PDMS wells while the dilution team prepared the microscope. To prepare the bilayers 30 µL of vesicle solution 3 (93.5 mol% POPC / 1.5 mol% PEG-DPPE / 5 mol% biotin- cap-DPPE vesicle solution) was put into 6 of the PDMS wells and 30 µL of vesicle solution 4 …show more content…
The microscope only needed to be focused the only light used for this experiment came from the microscope itself and not an external laser. The 4 PDMS wells were cleaned the same way as previously described. This time the glass coverslips was scratched with a diamond etcher before sticking the well to it. Two of the wells were then filled with 30 µL of vesicle solution 1 (contains: 99.5 mol% POPC with 0.5 mol% Texas Red labeled DHPE) and the other two with vesicle solution 2 (contains: 69.5 mol% POPC, 30 mol% cholesterol, and 0.5 mol% Texas Red labeled DHPE). These were then incubated for 30 minutes. After incubation the 30 µL of vesicle solution were suctioned out of the wells and the wells were refilled with 30 µL of PBS buffer. The wells were then sealed with another glass coverslip. The samples were flipped over before they were viewed so the bilayer would be on the top of the well. The microscope was focused and the sample was viewed with the 40x objective. We began photobleaching the samples at this point; they were photobleached with a green light at 100% intensity. We photobleached the sample without cholesterol for 3 minutes, we then took the light intensity down to 10% to take images of the sample and shuttered the light after each picture to prevent further photobleaching of the sample. For the first 10 minutes a photo was