This method is useful for answering certain key questions in the field of photosynthesis research such as are putative thylakoid complexes physiologically real complexes with energetically connected components? The main advantage of the green gel and TCSPC protocol described here is that it's quick and robust. Samples can easily be processed and analyzed in a single day, making issues of storage and stability less of a concern.
Though TCSPC can provide insight into the photosynthetic systems that we've studied here, it can also be applied to other systems ranging from chemical through physical and biological such as studies of fluorescent molecular motion in liquids, cells, or novel-structural motifs such as monomolecular layers and ionic liquids. To begin this procedure, prepare the needed stock solutions and green mini gels as outlined in the text protocol. Next, obtain some ice and dim the lights.
Using a glass Dounce homogenizer, completely homogenize the spinach leaves in approximately one to two milliliters of TMK buffer. To create a simple filtering device, cut a delicate task wipe in half and fold it into quarters. Remove the plunger from a syringe, and then pack the folded task wipe into the bottom of the syringe.
Pipette the leaf homogenate into the center of the task-wipe filter, then use the plunger to pass the homogenate through the filter and collect it in a 1.5-milliliter centrifuge tube. Centrifuge the homogenate at 5, 000 g and four degrees Celsius for 10 minutes to pellet the insoluble material, including the thylakoid membranes. Discard the supernatant and resuspend the pellet in one milliliter of TMK buffer.
To extract chlorophyll from each sample, take a 50-microliter aliquot and transfer it into a 1.5-milliliter microcentrifuge tube. Add 950 microliters of methanol, cap the tube, and mix by inverting it several times. Centrifuge at 10, 000 g for 10 minutes to pellet the precipitated proteins.
Using the chlorophyll concentration measurements as a guide, remove and discard some volume from each sample as necessary until each tube contains the same total amount of chlorophyll. Centrifuge at 5, 000 g for 10 minutes to re-pellet the thylakoid membranes. Remove and discard the supernatant, being careful to remove all of the supernatant without disturbing the pellet.
First, thaw an aliquot of TMK 30%glycerol detergent solution. Invert several times to mix and keep the solution on ice. Add an appropriate volume of the detergent solution to dissolve the pellet and result in a final chlorophyll concentration of one milligram per milliliter.
Pipette up and down repeatedly while being careful to avoid frothing in the sample. Then, keep the sample on ice to allow thylakoid samples to solubilize. After solubilization is complete, centrifuge the sample at 10, 000 g and four degrees Celsius for 10 minutes to pellet the insoluble material, resulting in a clear, dark green supernatant.
After this, load 15 microliters of solubilized thylakoid supernatant directly onto each well of a previously prepared 1.5-millimeter native gel. Using 1X running buffer, begin running the native green gel at 100 volts. Place the entire gel tank on wet ice for the duration of the run to mitigate resistive heating.
When the gel has finished running, remove it from the electrophoresis cell and rinse the running buffer off of the gel plates with distilled water. Remove the top plate from the gel and then rinse the gel with distilled water. Place the gel, which is still on the bottom glass plate, on ice;when the gel is not in use, cover it with plastic wrap and keep it in the dark to prevent it from drying out.
Next, use a sharp scalpel or razor blade to excise each band of interest when ready to proceed with the TCSPC analysis. Make sure that the excised band contains no contaminating band material. For each complex being analyzed, use a fluorescent spectrometer to make a room-temperature fluorescent spectrum between 600 and 800 nanometers.
Place masking tape on either end of the slides and fold it over several times to create spacers, which will allow the slides to be held together firmly without compressing the space between, then sandwich the gel slice between two glass microscopy slides. Add a small amount of water to the gel slice at the edge of the glass slides to create a smooth interface that will reduce signal scattering. Next, clamp the gel sandwich in the beam path so that the beam strikes the gel slice through the open edge of the plates.
For each complex, collect 10, 000 total data points at regular intervals across the fluorescence emission spectrum. To analyze the data for a given complex, first normalize the peak height of each decay curve for all of the wavelengths collected, then tail match each decay curve to the steady state fluorescence spectrum of the complex as outlined in the text protocol. Representative results for the green gel electrophoresis provides an example of an ideal result for the analysis of spinach thylakoids, where a number of clear, sharp green bands are visible.
Lanes 2 and 3 present more typical results from simple thylakoid isolation and electrophoresis on a non-gradient 5%acrylamide gel. Lanes 4, 5, and 6 provide an example of poor results due to increasing degrees of under solubilization of the thylakoid sample, while lane 7 demonstrates the results that can be achieved when Arabidopsis thylakoids are used instead of spinach. After the data curves of the TCSPC data have been set to the same time register, they can be normalized to the same peak height, which allows for the curves to be compared as a first analysis of the data.
Peak-normalized decay curves from different complexes can then be overlaid with one another at a given wavelength, allowing the differences in behavior between the complexes to be visualized. Tail matching the decay curves allows for the construction of decay-associated spectra. As seen here, the decay-associated spectra for the LHCII is notable for the lack of dynamic features and the shape of the LHCII fluorescence spectrum remains the same as the signal decays over time.
The decay of the fluorescence spectrum is also delayed, requiring 100 picoseconds to reach maximum fluorescence. This suggests that energy is not transferred between energetically distinct pigments within the complex as the fluorescence decays. The decay-associated spectra is then constructed for the Band 5 complex.
Compared to the LHCII, the fluorescence from Band 5 decays much more rapidly, reaching maximum intensity in only 30 picoseconds and then decaying to less than 20%of the initial intensity after 500 picoseconds. While attempting this procedure, it's important to remember that interpretation of the TCSPC data will rely on additional biochemical information identifying the components of the complexes under study, for instance, by 2 DSDS-PAGE and Western Blotting or mass spectrometry.
