The overall goal of this procedure is to label cells expressing a tetra cysteine and fluorescently tagged protein with the dyes flash or ash, and to quantitatively analyze the results. This is accomplished by first transecting, a plasmid carrying a fluorescent protein and the target protein containing a tetra cysteine tag into the cell line of interest. Next, the expressed protein is labeled with R or flash, and the non-specifically bound diet is removed by washing with BAL to prepare for microscopic imaging.
Then a confocal microscope is used to collect fluorescent images of the cells with the ash or flash and fluorescent protein channels. Finally, the images are analyzed using image J to quantify binding of ash or flash to the target protein. Ultimately, results can be obtained that show the extent of ash or flash binding to the target protein in cells through the correlation of ash fluorescence with fluorescent protein fluorescence.
The main advantage of this technique over existing methods like simply taking the target protein with GFP, is that can track a second property of the protein, such as conformational change in addition to it's localization. This method can help answer key question in the protein substructure and function field, such as ization events, conformational changes, and LI binding events that alter the ability of the TAC to ban ash or flash. We first had the idea for this method when we wanted to assess how small submicroscopic polygamous of met Huntington formed in cells.
Visual demonstration of this method is critical as the data analysis step are difficult to learn because they are technically complex Demonstrating the experimental procedures. Steps one to three will be Yasmin Ranson, a research assistant from our laboratory, demonstrating the data analysis. Step four will be Sevki GaN, a grad student from our laboratory Using standard cell culture methods for your cell line of interest and a live cell imaging slide ready for transfection.
Prepare a culture of adherence cells transfect your plasmid containing the TC tag gene of interest. According to a transfection method of choice, It is important to have positive and negative controls so as to assess the extent of specific binding to the TC tags, as well as to assess for bleach flow of fluence between channels when collecting confocal micrographs. Therefore, for two colors, ensure samples are prepared for single colors, either the fluorescent protein by itself and if possible, a T ctech protein bound to flesh or vs.
But without a fluorescent protein. One to two days after transfection, gently rinse the cells with 300 microliters of pre-war Hank's balance salt solution or HBSS, prepare fresh prewarm HBSS with 10 micromolar, one, two, et Ethan dia thiol or EDT and one micromolar flash or ash. And after removing the HBSS from the cells, gently replace with 300 microliters of the ash medium incubate for exactly 30 minutes at 37 degrees Celsius in a tissue culture incubator.
In our experience, longer incubation times significantly increase the background fluorescence. Gently aspirate the labeling solution from the cells and replace it with 300 microliters of prewarm HBSS with 250 micromolar, two three dimer, cap propanol, or BAL for 15 minutes at 37 degrees Celsius. Remove the wash solution by gentle aspiration and replace it with 300 microliters of prewarm HBSS.
After this wash, the cells may be fixed with paraform height, although we found that this increases non-specific cynical fluorescence. Hence, we usually image the cells live at room temperature. In addition, fixation of cells before labeling prevents bial dye binding.
To begin imaging cells on the confocal microscope, set up parameters for imaging the individual fluorophores and ensure that there is negligible bleed through between the channels. Next, adjust the photo multiplier settings so that the maximum fluorescence in the sample does not saturate the detector. Once the best imaging settings are determined, do not change any of them between samples.
Use a scan rate of 200 hertz, collect four line averages, and set the pinhole diameter to one area unit. The pinhole diameter can be expanded if signal to noise is an issue. However, this may result in some loss of imaging resolution.
Collect the confocal images in a 12 bit or higher format if possible. To maximize the quality of quantitative data analysis. Collect images for the fluorescent protein channel and also for the BI arsenal die channel for all samples.
To analyze the data, use Image J software with the following plugins. If using a version of Image J older than version 1.32 C, click on the following options. To enable multiple images to be opened at one time, edit options input slash output, click the box to use j file chooser to open slash share.
Click okay. Open the images of interest. Image J will automatically define the maximum and minimum pixel intensities that are displayed on the screen for each image separately, because different images will have different pixel intensities.
This means the displayed images are not comparable as viewed. To ensure the opened images are all on the same scale, physically define the upper and lower pixel intensities to be viewed. To set these values, choose image, adjust brightness slash contrast.
Click on set, define the minimum and maximum displayed values. Then choose propagate to all open images and click okay. Alternatively, place all the images for a particular channel together into a single file or stack.
Image J will then automatically adjust all the images to the same scale. To do this, choose image stacks images to stack enter a name common to each file and choose used titles as labels. Click okay.
Convert all the open images to eight bit for analysis. By choosing image type eight bit, this will rescale the viewed range into a zero to 255 interval. It's important not to save over the original 12 bit or higher format as this will lose information content.
Save a copy in a new folder called eight bit converted using the save as Option. One method to examine the extent of bi arsal die binding in a whole image is to perform a pixel intensity correlation plot. This plot's every pixel value in one channel relative to the corresponding pixel value in a second channel.
Hence, a pixel position high in GFP fluorescence will also be high in ash fluorescence if there is high binding. To analyze the pixel co correlation, make sure that the eight bit REA and GFP images are open in image J.Next, open the image correlator plugin by choosing plugins image correlator. Assign the image files to image one and image two.
Then click okay. The resulting stack may not display any detailed information. This is normal.
Save the stack in a new folder called scatterplots and give it the same file name as the sample it refers to. For example, Ash GFP Correlation plot. To view the data, choose one of two options.
First, transform the data so that it is a log format by choosing process math log auto. Then to visually rescale the data, click image adjust brightness slash contrast and select auto. Second, rescale the data visually to only show low values and display the data using a special lookup table or LUT to create a custom LUT.
Click on image color edit LUT for a very simple LUT. Click on the upper left pixel and choose the color black. Then select the next pixel to the right and choose the color red.
Next, click on the third pixel, and while holding the mouse button, drag the selection to the lower right pixel. Select the color red, click okay, and then select the color white and click okay. Select the brightness slash contrast range from zero to 255 as described earlier to lock in the image to that shown on the screen.
Convert it to RGB format to copy and paste images to other programs. First, open the eight bit or 16 bit images as described earlier. Assign an LUT color scheme to the images for GFP.
Assign the green LUT by clicking on image. Look up tables green. Finally select the image to copy and copy it to the clipboard By choosing edit copy to system, this figure shows a typical result for the kinase sock fused, the GFP and a TC tag that has been stained for 30 minutes.
With ash in HEC 2 9 3 cells, it appears that ASH binds efficiently to the target protein carrying a TC tag with high affinity. In contrast, this figure shows the corresponding control sample of SARK fused to GFP, but lacking the TC tag, which is essential for verifying both the specificity of the TC tag and the level of background staining. This result shows that RH has no reactivity with the target protein, lacking the TC tag, which indicates the specificity of the TC tag for SSH binding.
While attempting this procedure, it is important to remember not to allow the cells to be confluent and calm, as this will impact V binding in cells. After watching this video, you should have a good understanding of how to simply analyze your data quantitatively, and you can refine these methods to develop your own unique IUTS for data visualization.
