Fluorescence Anisotropy Lab - Optical Spectroscopy | PHYS 552, Lab Reports of Optics

Download Fluorescence Anisotropy Lab - Optical Spectroscopy | PHYS 552 and more Lab Reports Optics in PDF only on Docsity! Physics 552 Optical Spectroscopy (Fall '08) - 1 - IN GENERAL, IF YOU DO NOT GET “GOOD” RESULTS FROM YOUR DATA, YOU SHOULD STILL ANALYZE/PRESENT YOUR OWN DATA, GIVE REASONABLE CONJECTURES AS TO WHY YOUR DATA COULD BE OFF, AND EXPLAIN WHAT YOU WOULD DO DIFFERENTLY TO TEST YOUR CONJECTURE. Fluorescence Anisotropy Lab Introduction In this lab, we will explore some aspects of fluorescence anisotropy. As will be discussed in lecture, excitation with polarized light will result in photoselection, i.e., favored excitation of fluorophores whose absorption moments are mostly aligned with the polarized light. Emission depolarization can occur for several reasons, including non-parallel absorption and emission transition moments, torsional vibations of the fluorophore, Brownian motion, and radiative and non-radiative transfer of excitation energy to another fluorophore with a different orientation. As such, fluorescence anisotropy measurements can be used to probe the size and shape of molecules, the rigidness of internal structure, as well as the viscosity of its environment. In this lab, we will focus on the effect of rotational diffusion on fluorescence anisotropy. For our measurements to be sensitive to rotational diffusion, we need the fluorescence lifetime and the rotational time constant to be comparable. In Experiment I, we will study the effect on the anisotropy as we increase the viscosity of the solution. In Experiment II, we use dynamic quenching to reduce the fluorescence lifetime of the fluorophore, so that it has less time to rotate. Finally in Experiment III, we will use anisotropy to measure the rotational diffusion and hence the apparent molecular volumes of two different proteins with tryptophan in them. References • Dean Eckhoff’s anisotropy notes • 2007 Lecture 23 – Fluorescence Anisotropy (linked in pdf version) • Valeur, Chapter 5 • Lakowicz, Chapter 10 Physics 552 Optical Spectroscopy (Fall '08) - 2 - Experiment I –Rotational Diffusion Effects on Anisotropy For the first part of the lab, we will study the effect of rotational diffusion on fluorescence anisotropy by changing the solvent viscosity. We will measure the fluorescence anisotropy of fluorescein in varying amounts of glycerol. 1. Make a manual measurement of the G-factor. G=IHV/IHH a. Place sample Gly0 in the sample chamber. b. Select the Instrument Control tab and change the settings to those shown: (Excitation at 480nm, polarizer at 90 deg on shutter open, Emission at 514 nm, 0 deg and shutter open). You can right-click on objects to change parameters. This will give you IHV. Record the intensity of the R.Em. PMT to two significant digits. c. Now change the emission polarizer to 90 deg and measure IHH. IHV IHH G 2. Measure the anisotropy for each of the 6 solutions. (See page 6 for general directions). a. Fluorescein settings: Excitation at 480nm, polarizer Emission at 514 nm, number of iterations = 40. b. Detect G-Factor once for each sample. Sample Glycerol Wt % ρ (g/mL) η/η0 rave Gly0 0 1 1.000 Gly1 25 1.059 2.0559 Gly2 48 1.12 5.4022 Gly3 64 1.1643 13.6297 Gly4 80 1.2085 59.78044 Gly5 96 1.2508 778.9 Physics 552 Optical Spectroscopy (Fall '08) - 5 - Experiment III – Anisotropy Application – Protein Rotation Here we will use steady-state anisotropy to measure the apparent molecular volume of two proteins: lysozyme, which we used in Lab 5, and bovine serum albumin (BSA), a protein used as a carrier of non- water soluble molecules in the bloodstream. Both proteins contain the fluorescent amino acid tryptophan, whose fluorescence we will be measuring. 1. Measure the anisotropy for each of the proteins. (See page 6 for general directions). a. Tryptophan settings: Excitation at 280nm, polarizer Emission at 335 nm, number of iterations = 40. b. Detect G-Factor once for each sample. Analysis We will use the Perrin equation and the equation relating the rotational constant to the MW of the protein: h)MW( +== νηηθ RTRT V where ν is the specific volume (~0.73mL/g) and h the hydration of the protein (~0.23 g water/ g protein) You will also need the values r0 = 0.26 for tryptophan, γ = 2 (average for proteins), MW of BSA = 66,400 g/mol, MW of Lysozyme = 14,300 g/mol. Q7) First assume that the fluorescence lifetimes of the tryptophans are the same in the two proteins and derive a relationship relating the anisotropies and MW’s. The equation should contain only r0 and the r and MW for the two proteins. Plug in the values and show that this equation is no good. Q8) Now, redo the calculation without assuming equivalent lifetimes. Use the relationship to determine the average lifetime of tryptophan fluorescence in Lysozyme assuming a lifetime in BSA of ~6.0 ns. *** Note that the fluorescence lifetimes of tryptophan are highly dependent on the local environment. Often times, even a protein with only one tryptophan residue (e.g. HSA) will have multiple lifetimes corresponding to different conformational states of the protein. In our case, BSA has 2 tryptophans and Lysozyme has 6, so it will be even more complicated. The single value we report will be the intensity- weighted average of these lifetimes. Physics 552 Optical Spectroscopy (Fall '08) - 6 - Again recall how to use the fluorometer: c. Check with your instructor that the monochromators have been calibrated. d. Make sure the POLARIZERS ARE IN, and that you are using the 1 mm slits e. In the Vinci software, enter in the global settings: i. Select from the menu bar Settings>>Global Settings… and enter the output directory. Use d:\users\P552\Lab06\<Day><Time> where, e.g., <Day><Time> is Thur1-4. ii. In the same dialog box, check that Prompt for experiment file name is checked. 1. We will be measuring anisotropy at a single wavelength and averaging over 40 iterations to get a good signal. Each sample will take a few minutes to measure. From the menu bar, go to Experiment>>Single Point>>Polarization. You should get the screen below: 2. From the menu bar select View>>Visualization. The Y axis should be Anisotropy and the secondary Y axis should be Total Intensity. 3. Set the excitation wavelength, emission wavelength, and number of iterations appropriately for each sample.