GFPタグTMEM184Aを使用すると、ヘパリン受容体のアイデンティティの確認のために構築します

The overall goal of using a GFP-tagged construct is to confirm the identity of a previously unknown protein using the GFP as a handle. This method can help with the functional identification of orphan genes when a specific enzyme activity is not available for confirmation. The main advantage of this technique is that the GPF-tag allows the same construct to be used for several types of experiments.

Generally, individuals who are new to this method may have trouble, because the transfection does not always result in the strong-construct expression, and the cells do not always revive. We first had the idea for this method, when we began to recognize the difficulties of identifying a protein using only antibodies created against it. Begin by resuspending the cells of interest in one milliliter of HEPES-buffered saline electroporation buffer and immediately place the cells on ice.

Add a sufficient volume of GFP-TMEM184A-tagged plasma to the cells to achieve a final concentration of 20 micrograms per milliliter of DNA, and transfer approximately 0.4 milliliters of cells into a pre-chilled electroporation cuvette. Electroporate the cells. Then see the transfected cells onto tissue culture dishes, and place the dishes in a cell culture incubator for 24 to 72 hours.

The transfection step is the most critical step of the procedure, because most of the remaining experimental work cannot be effectively completed if the cells are not viable, and do not express significant levels of the GFP-tagged construct. At the appropriate experimental time point, add 100 micrograms per milliliter of Rhodamine Heparin to the GFP-plasmid-expressing cell culture medium, and incubate the cells, protected from light, until the desired level of staining is reached as observed by fluorescence microscopy. Next, rinse the cells with PBS and a few seconds of gentle shaking, protected from light, followed by fixation with 4%paraformaldehyde for 15 minutes at room tempurature with gentle shaking.

To assess the GFP-tagged Rhodamine Heparin co-localization, use a confocal microscope with the appropriate GFP and Rhodamine excitation and diminished lasers to image at least 50 cells from each separate labeling experiment. Examine the images in the appropriate image-analysis program to determine the relative Rhodamine finding and uptake in each cell, using a free-hand tool to circle the cells within each fluorescent image. Use the measure tool to determine the intensity within each circled space, then export the fluorescence-intensity data to a spreadsheet to calculate the area multiplied by the intensity, subtracting the background for the same amount of area for each cell.

The fluorescence per cell can then be averaged for all of the cells, to obtain statistically significant data. To facilitate the fluorescence resonance energy transfer from GFP TMEM184A to Rhodamine Heparin, first mount fixed GFP TMEM184A transfected Rhodamine Heparin-labeled cells onto glass microscope slides. Excite the cells at 405 nanometers for imaging the Rhodamine emission, followed by excitation of 488 nanometers to image the GFP emission.

Then excite the cells at 561 nanometers for a second Rhodamine emission imaging. For live-cell imaging of the Rhodamine Heparin uptake, seed a 100-milliliter confluent dish culture of the GFP TMEM184A-transfected cells into 635 millimeter live-cell imaging dishes to obtain cells near confluence. After 48 hours, replace the supernatant with culture medium without Phenol red, and transfer a dish of cells to a confocal microscope with a warming stage set to 37 degrees Celsius.

Focus the microscope, and add 100 micrograms per milliliter of Rhodamine Heparin to the cells with gentle mixing. Then immediately begin recording the live images. If no Heparin uptake occurs within the field of view, move the dish slightly to identify the cells with at least one green vesicle, containing the Rhodamine label.

For Heparin-binding assessment in vitro, rinse one black ativan-coated 96 well plate and one black non-coated 96 well plate with 200 microliters of 0.2%CHAPS PBS pro-well for three five-minute washes with shaking. After the last wash, add 100 microliters of 60 picomoles per well of biotinylated anti-GFP in triplicate to the ativan-coated plate for each GFP finding group. Next, add buffer only to the control wells and seal all of the wells with a plate cover for a two-hour incubation at room tempurature with shaking.

At the end of the incubation, wash all of the wells three times in CHAPS PBS, as just demonstrated, and add 100 microliters of five nanomoles per well of GFP TMEM184A, or GFP alone, to the appropriate wells in the ativan-coated plate. After a one-hour incubation at room tempurature with shaking, wash all of the wells with fresh CHAPS PBS, and add 100 microliters of each experimental fluorescein-Heparin concentration, to the appropriate test wells in triplicate, to the ativan-coated plate. Add the concentration standards to both the ativan-coated and non-coated plates, and incubate both plates for 10 minutes at room tempurature, with shaking, protected from light.

Now, use a plate reader to record the initial Heparin-fluorescence emission in the control wells of both plates, and the initial total fluorescence emission from the wells with the immobilized GFP or GFP TMEM184A in the ativan-coated plate. Then transfer the unbound fluorescent Heparin from the wells with the immobilized GFP TMEM184A or GFP into the corresponding wells in the non-coated black plate. After immediately reading the fluorescence emission on the non-coated black plate, add 100 microliters of fresh CHAPS PBS back into wells from which the fluorescein-Heparin was removed, and read the fluorescence emissions for these wells to obtain the fluorescence emission from the removed, unbound samples in the non-coated plate.

When all of the unbound readings have been obtained, use the total Heparin emission values to confirm the correct levels of Heparin were added to each well, by subtracting the average background readings from the values. Then create a plot of the emission versus the total fluorescein-Heparin added, to determine the scale of the emission versus the amount of Heparin, and plot the corrected bound Heparin, versus the added Heparin for the triplicates from both of the GFP and GFP TMEM184A wells. The length of time after transfection impacts the distribution of the GFP TMEM184A, which appears to accumulate over at least 72 hours, with more GFP apparent in the paranuclear region over time.

The staining pattern of GFP in this representative vascular cell culture, appears to be most similar to that of an antibody raised against a C-terminal peptide, a logical outcome, given that the GFP is at the C-terminus of the protein. The use of a fluorescein-Rhodamine-Heparin ligand can also be used for the real-time evaluation of ligand receptor co-localization over time. Heparin uptake is increased in GFP TMEM184A construct transfected stable knockdown cells, resulting in cells that can regain the ability to internalize Rhodamine Heparin at or above the level of wild-type cells.

Using the demonstrated plasmid-encoded GFP tact system, the anti-GFP antibody isolated GFP TMEM184A construct demonstrates a specific satriple Heparin binding, while the control GFP does not bind any Heparin. Simple immunofluorescence staining with antibodies again GFP can also provide information about the GFP location, based on the antibody access in non-permeabilized cells, providing preliminary evidence that the GFP expressed at the plasma membrane is extra-cellular. Once mastered, this technique can result in excellent GFP-construct expression, after 24 to 48 hours of culture.

While attempting the live imaging protocol, it is important to remember that some cells might not exhibit the co-localized uptake, so it is often necessary to examine other cells. Binding assays with GFP ligands can be complicated by dimer or higher-order aggregates of the GFP protein, particularly if they are in detergent micelles. Overall, the expression of GFP-tagged proteins can facilitate a wide variety of experiments to confirm the identity of a previously-unknown protein.