The overall goal of this procedure is to map sites of ACAP LBC interaction with PDE four D three. By measuring the mean fluorescence intensity of biomolecular, fluorescence, complementation or BC using flow cytometry. To do this, cells are transfected with ACAP LBC and PDE four D three fragments fused to the N or C terminal portion of the fluorescent reporter protein.
Venus Flow cytometry is then performed to assess fluorescence if the protein fragments interact Venus is completed and fluorescence. If the protein fragments do not interact, no fluorescence is detected. Complimentary western blood analysis is performed to determine the relative protein expression levels.
The strength of the protein protein interactions is then calculated by normalizing YFP mean fluorescence intensity with protein expression levels. The resulting data indicate that the binding of PDE 43 to ACAP LBC is mediated by multiple regions within PDE 43, primarily through upstream conserved region one or UCR one. The main advantages of this technique over existing method like co immunoprecipitation and BP C using microscope or that it is relative simple, it's sensitive and it facilitates the rapid screening of a large number of cells.
Though this method can provide insight into protein-protein interactions, it can also be used to screen for factors that regulate protein-protein interactions for drug discovery. Generally individuals new to this method will struggle because a linear B response needs to be determined. An expression of construct should be similar so that result use different protein fragments can be compared to one another.
In the experiments described here, the cells are cot transfected with N terminal fragment of the YFP derivative, Venus fused to full length ACAP LBC and the C terminal fragment of Venus fused to one of the following, full-length PDE E 43, the upstream conserved region one UCR one of PDE E 43 or the upstream conserved region two plus catalytic region UCR R two plus CAT of PDE 43 the day before transfection in six. Well, tissue culture plates seed 1.2 times 10 to the fifth HEC 2 9 3 T cells per well in two milliliters of complete growth. Medium seed, three control wells plus three wells for each of the test.
Cotran incubate at 5%carbon dioxide at 37 degrees Celsius overnight. The next day prepare for transfection of the BC plasma DNA constructs and CFP vector, which is cot transfected with BS C constructs as a marker. For each condition, add the appropriate amount of DNA to 250 microliters of Optum M1 serum free medium.
In a sterile tube, one tube is enough to transfect three wells. Then add three microliters of transit LT one reagent for each microgram to the diluted DNA mixture and pipette gently to mix incubate for 30 minutes at room temperature. Following the incubation at 250 microliters of transfection mixture dropwise to the cells then incubate at 5%carbon dioxide at 37 degrees Celsius, 24 hours.
Post transfection check fluorescence under an epi fluorescence microscope, the cells should express yellow fluorescence for BC signal and CFP fluorescence for reporting transfection efficiency. To prepare the cells for flow cytometric analysis first wash them with two milliliters of ice cold PBS then detach the cells by adding 250 microliters of 0.05%Trypsin incubate for two to five minutes at 37 degrees Celsius. Then using a microscope check for cell detachment.
Once the cells have detached, neutralize the trypsin with one milliliter of DMEM. Next, transfer one milliliter of cells to one micro centrifuge tube, then transfer 0.25 milliliters of cells to a second micro centrifuge tube. Spin the tubes at 500 cheeses for five minutes.
The 250 microliter sample will be further processed for flow cytometry. Set the one milliliter sample aside on ice for western blood analysis, which should be performed in parallel following the spin Resus. Suspend the cells in 250 microliters of fax buffer and place them on ice cells can also be fixed in 1%Paraform aldehyde in PBS If flow cytometry analysis will not be immediate here, flow cytometry is performed using a Beckman Coulter cyan a DP equipped with 488 nanometer 405 nanometer and 642 nanometer solid state lasers summit software is used for acquisition and analysis after calibrating the flow cytometer using standard beads, plot forward scatter or FSC versus sideward scatter SSC on a linear scale and gait on the live cell population.
Next plot SSC versus the voltage pulse width and gait on the non aggregated live cell population. Then plot FSC on a linear scale versus CFP fluorescence at 405 FL six on this instrument on a log scale and gait on the non aggregated live CFP positive cells. Finally, plot FSC on a linear scale versus YFP fluorescence at 488 nanometers FL one on this instrument on a log scale to visualize BC intensity.
Next, run the Y-F-P-C-F-P double negative sample and adjust the F-S-C-S-S-C FL one and FL six photo multiplier tube or PMT voltage to set the threshold for each signal. Next, run both YFP and CFP single positive samples for future offline compensation. Be sure to acquire at least 10, 000 gated events.
Finally, collect data for experimental samples following data collection. Set up the analysis protocol as per acquisition with propagating with gate one in FS CSC dot plot for live cells. Gate two in FSC pulse with dot plot of gate one for non aggregated live cells and gate three in ccfp FSC dot plot of gate two for non aggregated ccfp positive live cells.
After compensating for YFP and CCFP signal in Y fp CFP dot plot using YFP and CFP single positive cells, we determine the cutoff lines for CFP and YFP positive cells. Export the YFP mean fluorescence intensity or MFI of CFP positive cells to excel for data plotting then in Excel normalize Y-F-P-M-F-I to the expression level of ACAP LBC PDE E 43 and loading control alpha tubulin as determined by western blotting to determine the linear range of YFP mean fluorescence intensity. CFP was cot transected with VN ACAP L BBC and varying amounts of VC PDE E 43 in HEC 2 93 cells and interaction was assessed using bif flow cytometry as described in this video, this plot shows relative fold change in expression of VC PDE E 43 as a function of VC PDE E 43 transfected.
The mean fluorescence intensity of Venus in CFP positive cells indicated by the blue squares correlated with VCDE 43 expression and was consistent with the expression levels determined by image analysis of a western blot, which is indicated by the red triangles. Similar CFP mean fluorescence intensity was seen among samples as indicated by the green squares and was used as a negative control to limit cell variations. These results validate the use of flow cytometry to quantify ACAB LB C PDE 43 interaction by measuring the mean fluorescence intensity of BS e while at the same time determining the proper amount of BIC constructs used for screening to map ACAP LBC binding sites within PDE E 43 BIC analysis of ACAP LBC interaction with full length PDE E 43 fl.
The UCR one region of PDE 43 and UCR two and CAT regions of PDE 43 was assessed using this method as seen here. Expression of vn ACAP LBC and VC PDE 43 UCR two plus CAT results in lower BC signal when compared to VC PDE 43 FL and VC PDE 43 UCR one similar protein expression was observed in all flow samples and mean fluorescence was normalized to the relative expression levels of ACAP LBC PDE 43 and alpha tubulin. Therefore normalized B-C-M-F-I indicates that there is no difference in the fluorescence intensity between ACAP LBC PDE 43 FL and ACAP LBC PDE 43 UCR R one, but much lower signal for ACAP LBC PDE 43 UCR two plus CAT interaction.
These results suggest that there are multiple regions in PDE 43 that interact with ACAP LBC and that PDE 43 UCR R one is the primary region of interaction with ACAP LBC. After watching this video, you should have a good understanding of how to study protein, protein interaction by flow cytometry analysis of BP C.While attempting this procedure, it is important to remember to determine the correct amount of BP C construct used to obtain similar protein expression and linear BP C response. Following this procedure, other methods like peptide or risk can be performed in order to determine which amino acids within PDE 40 D three are responsible for.
Its ACAP RBC interactions.
