This experiment aims to define whether two proteins interact in cells and to delineate aspects of the sites of that interaction. First cells are transfected with expression constructs encoding the two proteins of interest, one of which is fused to the end terminus of a fragmented fluorescent protein and the other fused to the complementary C terminus of the fragmented fluorescent protein. After allowing the transfected cells to develop fluorescent signal in culture, the protein complexes of visualized by imaging and immuno blotting.
Next, the collected images are exported to imaging software such as Image J In order to quantify average fluorescence intensity per cell in A controlled experiment, BIFC results can demonstrate protein protein interactions and the localization of these complexes, like the SH three domains of the scaffold protein intersecting and the pro rich domain of PI three KC two beta. The main advantage of this technique over existing methods such as colocalization, immunoprecipitation, and fret, is that BFC allows for the spatialized localization of weaker transient interactions in the cell. And this method does not require extensive post-processing images as seen with fret.
This method can address key questions in the area of signal transduction, such as DO proteins X and Y interact, and if so, what are the specific compartments to which these complexes localized in the cell? Generally, newcomers to this method will struggle because they haven't chosen the proper configuration for expressing their biopsy. Tagged proteins of interest Begin by selecting one of the multiple fluorophores for the BIFC fusion proteins.
Consider the amino and carboxy terminal ends of Venus are able to form a complex at 37 degrees Celsius while the Y-F-P-B-I-F-C fragments require a pre incubation at 30 degrees Celsius. Design expression vectors for tag addition to the proteins of interest by considering how attachment of the BIFC fragments may affect the function of the proteins of interest. For example, RAs family, GTP a's are lipid modified at the carboxy terminus and cannot be tagged at this end without disrupting this modification.
Always perform pilot experiments to determine transfection conditions. Also monitor the cells by fluorescence microscopy to identify an optimal time for signal development. Then perform western blossom analysis to confirm equal expression of the constructs.
Importantly immuno stain for the HA or flag tag to verify that the addition of the BIFC fragments do not alter cellular localization of the proteins of interest for each sample plate. 1.3 times 10 of the five cause cells each onto one glass bottom matter plate and two wells of a six well plate and allow the cells to settle overnight in a 37 degrees Celsius incubator the next day. Prepare DNAs for transfection by lipectomy or other preferred method.
First, dilute the DNA in 250 microliters of serum free DMEM as a transfection control. Add CFP at one 50, the amount of your total BIFC DNAs. Now dilute the lip in 250 microliters of serum free DMEM using 10 microliters of lip per one microgram of DNA.
Mix the DNA and lip dilutions incubate at room temperature for 20 minutes. Rinse cells twice with warm serum free DMEM. Add two milliliters of serum free DMEM to each glass bottom plate or well of a six well dish.
Then split each transfection mixture evenly between one glass bottom dish and two wells of a six. Well plate incubate the cells at 37 degrees Celsius for five hours. Finally, remove the transfection media and replace with complete DMEM plus 10%FBS media.
Incubate the cells for the time determined in pilot experiments to obtain the necessary expression levels of the proteins of interest. Examine the transfected cells under an epi fluorescent microscope to ensure that the positive control is fluorescent. Rinse the cells three times with PBS for live cell imaging.
Place the cells in media lacking indicator dies. For imaging fixed cells. Add 2%para formaldehyde and place on ice for 10 minutes.
Then rinse the cells three times with PBS now covered with one milliliter of PBS and store in the dark at four degrees Celsius, immediately lyce the cells in the six well dish. For Western blot analyses, it is important that all cells are prepared at the same time so that the lys cells are representative of the image cells. Perform a western blot to ensure that the tag proteins are expressed at equivalent levels.
When imaging cells with a confocal microscope, it is important to keep settings constant throughout the experiment so that the fluorescence is comparable between samples. Be sure to image individual cells where pixels are not saturated. Since CFP was included as a transfection control, select only CFP positive cells for analysis of BIFC signals.
Quantify fluorescence using imaging software such as image J.Open image files in Image J.Go to analyze set measurements. Check the boxes for area and mean gray value in the measurements box. Note that in this example, venous images have been pseudo colored green and CFP images, pseudo colored red.
Using the freehand selection tool, draw an outline around the edge of the entire cell in the CFP channel leaving this outline in place. Shift to the YFP channel. Go to analyze, measure the mean gray value reflects the average fluorescence intensity per cell for each image.
Draw a circle in the YFP channel in an area that does not contain a cell. Take a measurement for this area as background. Subtract the background from each image.
Finally, to calculate average fluorescence intensity for a population of cells, average the mean grade value minus the background for all the cells imaged in one sample. Ideally quantify 60 cells over three experiments. The multi-domain scaffolding protein intersecting regulates neuronal survival through regulation of a novel class two PI three kinase, PI three KC two beta intersecting SH three domains interact with the amino terminal proline-rich region of PI three KC two beta transfection of VN tagged intersecting with VC tagged PI three KC two beta results in a fluorescent signal in the YFP channel, which is pseudo colored green indicating complex formation.
CFP pseudo colored here in red is used as a control to mark transfected cells mutations in the proline-rich domain of PI three KC two beta, which disrupt co precipitation of intersecting and PI three KC two beta decrease the BIFC signal. This difference in BIFC signal is not due to differences in expression of the wild type and PA PI three K proteins Once mastered, this technique can be performed in three days if done properly. While performing this procedure, you need to remember to run the proper controls and to check for protein expression to ensure that differences in BC signal are due to differences in complex formation and not due to expression following this procedure.
Other techniques such as surface plasma on residents can be performed in order to answer questions such as what are the different affinities of these complexes for each other?
