A Fluorescence Mthod to Study BSA Protein Hydrogels
Mentor 1
Ionel Popa
Location
Union Wisconsin Room
Start Date
5-4-2019 1:30 PM
End Date
5-4-2019 3:30 PM
Description
Here we want to develop a new method of measuring the mechanical response of biomaterials in parallel with a fluorescent probe, which reports on the amount of secondary structure inside the protein hydrogel. Low volume Bovine Serum Albumin (BSA) is turned into hydrogels of cylindrical shapes using Teflon tubes as a template. A hydrogel is tethered between two hooks connected to a voice coil motor and a force sensor. An analog Proportional Integral Differential (PID) system compares the force measured by the force sensor with the setpoint from the computer and adjusts the gel extension by moving the voice coil to minimize the difference between the two inputs. This clamping of the force allows for new types of experiments for measuring the biomechanics of proteins. By using an inverted microscope and an EMCCD camera, we can measure the fluorescence signal coming from the fluid chamber to quantify the domains unfolding.
With the use of 1-anilino-8-naphthalene sulphonate (ANS), a fluorescent marker, we can decouple the extension from unfolding protein domains and the matrix shifting. ANS binds to the hydrophobic parts of the molecule and emits a fluorescence signal when it is excited at 350-nanometers. As the protein is stretched on the Force Clamp Rheometer, the molecule loses its fluorescence due to losing the hydrophobic zones which decreases the amount of bound ANS. This method will allow us to use a bulk approach to measure the mechanical properties of single molecules which we are doing now using magnetic tweezers. This way we will also be able to answer the question of how proteins behave in crowded environments and how they unfold inside of hydrogel.
A Fluorescence Mthod to Study BSA Protein Hydrogels
Union Wisconsin Room
Here we want to develop a new method of measuring the mechanical response of biomaterials in parallel with a fluorescent probe, which reports on the amount of secondary structure inside the protein hydrogel. Low volume Bovine Serum Albumin (BSA) is turned into hydrogels of cylindrical shapes using Teflon tubes as a template. A hydrogel is tethered between two hooks connected to a voice coil motor and a force sensor. An analog Proportional Integral Differential (PID) system compares the force measured by the force sensor with the setpoint from the computer and adjusts the gel extension by moving the voice coil to minimize the difference between the two inputs. This clamping of the force allows for new types of experiments for measuring the biomechanics of proteins. By using an inverted microscope and an EMCCD camera, we can measure the fluorescence signal coming from the fluid chamber to quantify the domains unfolding.
With the use of 1-anilino-8-naphthalene sulphonate (ANS), a fluorescent marker, we can decouple the extension from unfolding protein domains and the matrix shifting. ANS binds to the hydrophobic parts of the molecule and emits a fluorescence signal when it is excited at 350-nanometers. As the protein is stretched on the Force Clamp Rheometer, the molecule loses its fluorescence due to losing the hydrophobic zones which decreases the amount of bound ANS. This method will allow us to use a bulk approach to measure the mechanical properties of single molecules which we are doing now using magnetic tweezers. This way we will also be able to answer the question of how proteins behave in crowded environments and how they unfold inside of hydrogel.
