The overall goal of this procedure is to determine the mobility of enhanced green fluorescence protein by measuring the frap of the protein in a region of interest. This is accomplished by first transecting, the EGFP vector in cultured rat hippocampal neurons. Next Fre recording is performed in a spine of interest using a Zeiss seven 10 confocal microscope.
The photobleaching rate can then be calculated using Image J and GraphPad Prism software. Ultimately, results can be obtained that show the mobility of EGFP in a spine from a cultured hippocampal neuron through confocal microscopy. This protocol was developed by Chazen in collaboration with myself, Ronald Pet, as well as Ya, Wang Bahar Kisha, and the senior author of the original paper, Robert Ol, who died in October, 2009.
We dedicate this video to his memory. This method can help answer key questions in the protein dynamics field, such as turnover rate of a protein of interest in a region of interest. Begin this procedure by culturing embryonic day 18 rat hippocampal neurons on poly de lysine coated mattec 35 millimeter glass bottom dishes on 16 to 18 days in vitro transfect neurons using the clone tech Cal Foss mammalian transfection kit before beginning the transfection.
Save the original culture medium in a sterile 15 milliliter tube for later use. Fill each 35 millimeter dish with 1.5 milliliters of DOL eco's modified eagle medium 30 minutes prior to transfection. Next, combine 10 micrograms of P-E-G-F-P-N one plasma DNA with 12.4 microliters of two molar calcium solution and bring the total volume up to 100 microliters with sterile water.
Add the DNA mixture DROPWISE to 100 microliters of two x heaps buffered saline. Vortexing the buffer after each drop is added. After letting the final mixture sit at room temperature for 20 minutes, add the buffer DNA solution to the DMEM incubated neurons.
Incubate the neurons at 37 degrees Celsius for one to one and a half hours. Following incubation, remove the calcium phosphate containing medium from the culture dishes and wash the cells with DMEM. Repeating the DMEM wash a total of three times.
Exchange the DMEM medium with the original culture medium before returning them to the incubator. Neurons are used for the frack experiment two to four days after transfection. To begin, replace the culture medium in the 35 millimeter glass bottom dish immediately with Prewarm Tyro solution.
A Zeiss LSM seven 10 confocal microscope is used for the frack experiment. The Zeiss temp module system maintains the temperature, the humidity, and the percent carbon dioxide of the working system. Make sure that the carbon dioxide tank is connected and that the water bottle, which is used for balancing humidity is filled with water.
Once the microscope is set up, find a transfected, mature dendrite with the 100 x objective. If the transfected cells in the dish are sparse, search for a desired cell with the 40 x objective. Then switch to the 100 x objective.
To capture images, use five x optical zoom and a 2 56 by 2 56 pixel resolution. To image a short piece of dendrite with several spines. To capture images, use nominal speed nine, which takes 0.5 seconds.
To finish a scan, set the pinhole to two micrometers to obtain strong fluorescence. When taking images, try to use low laser transmission, for example, one to 5%to avoid photobleaching the entire image. Next, select the spine of interest for this experiment.
Mushroom spines with spine head diameters of about one micrometer are chosen to perform a wrap experiment. Take five control images before bleaching, then bleach the spine of interest 10 times at nominal 100%Laser transmission capture a series of images immediately after bleaching. The interval of time should be adjusted according to different targeting proteins and different experimental designs.
For this experiment, images are captured every second for 15 seconds after bleaching. Next, save the images to analyze the data. Open images with Image J software align the stack of images using the aligned tool so that the spine of interest does not float and remains in the same position.
On the image measure relative fluorescence intensity of the spine of interest, which is a transfected but unbleached region. Measure the background region in time-lapse images with the intensity versus time monitor tool of image J.Use a non fluorescent region as the background region curve fit the fluorescent intensity with a one phase exponential equation in GraphPad Prism software or other similar software. Finally, calculate the percent mobile and immobile fraction of fluorescence.Shown.
Here are the frack measurements of EGFP fluorescence in a spine from a cultured hippocampal neuron. The region of the spine control and background are marked, and the red arrowhead indicate the time of photo bleaching. Photographs of the same area were taken before and at 0 1, 5, 10, and 15 seconds after photo bleaching.
FRA curves of EGFP fluorescence are shown over a 15 second period with the dots on the curves representing the FRA at every second. The curves were fitted by one phase exponential equations. The average fluorescence before photobleaching was counted as 100%In this experiment, the mobile fraction is 87%and the immobile fraction is 13%A.
After watching this video, you should have a good understanding of how to measure the mobility of A GFP tagged protein by using FRAPPE technique.
