The overall goal of the following experiment is to observe the interaction between two proteins and live cultured cells. This is achieved by sub cloning the CDNAs for the two proteins of interest into plasmids containing the coating sequence for luciferase or yellow fluorescent protein, abbreviated YFP to create vectors for the expression of protein in mammalian cells. As a second step, the plasmids are transfected into cultured cells, which leads to the expression of YFP and luciferase fusion proteins.
Next luciferase substrate is added to the cells in order to initiate light emission from the luciferase. Results are obtained that show interaction between the two fusion proteins based on energy transfer from LUCIFERASE to YFP. The main advantage of this technique over existing methods like coun precipitation, is that it allows protein protein interactions to be observed in live cells.
This Method can help answer key questions in the field of functional genomics by determining if a mutation found in a patient affects protein protein interactions. Demonstrating the first step of the procedure will bes RAB tric, a PhD student from my laboratory To begin create the plasmids as described in the text protocol. Estimate the concentration of them based on absorbance at 260 nanometers.
Determine the molecular mass of each plasmid by multiplying the number of base pairs by 650.Daltons. Use the concentration and molecular mass to calculate the molar concentration of each plasmid preparation. Dilute the plasmid DNA preparations to a concentration of 36 nano molar.
These will be the working stocks that will be used to prepare the DNA mixes. For transfection. The most difficult aspect of this procedure is setting up the DNA mixes To ensure success, we create spreadsheets to precalculate the correct amount of each component, and we take great care.
In setting up the DN mixes. Prepare the first control DNA mix containing 1800 nanograms of filler plasmid in a final volume of 20 microliters of water. Then set up a second controlled DNA mix by preparing five microliters of the P look control construct.
Add filler plasmid to bring the total DNA mass to 1800 nanograms. Add water to bring the final volume to 20 microliters. Prepare a third control DNA mix containing five microliters of P look control construct, and five microliters of PYFP control construct.
Add filler plasmid and water as before. Finally, set up a fourth control DNA mix containing five microliters of the positive control construct. Again, add filler, plasmid and water in the same manner.
Next, prepare 3D NA mixes to test for homodimerization of a protein of interest x. For each DNA mix, combine five microliters of the relevant P look construct and five microliters of the PYFP construct, adding filler plasmid and water. Then prepare DNA mixes to test for an interaction between a pair of proteins of interest X and Y.For each DNA mix, combine five microliters of the relevant P look construct and five microliters of the PYFP construct along with filler plasmid and water harvest sub confluent hec 2 9 3 cells from a 75 square centimeter flask dilute 10%of the total cells into 13 milliliters of culture medium.
Then dispense 130 microliters of cell suspension into each well of a white clear bottomed 96 well tissue culture plate before culturing the cells for 24 hours. Next, calculate the number of wells to be transfected by multiplying the number of DNA mixes by three. Prepare a master mix containing 6.3 microliters of room temperature, serum free medium, and 0.18 microliters of transfection reagent per well mixed by vortexing before incubating at room temperature for five minutes.
Then dispensed 20 microliters of serum free medium transfection reagent master mix into the required number of tubes. Add two microliters of the corresponding DNA mix into each tube, incubate at room temperature for 10 minutes. Finally, transfect three wells with each transfection mix.
Dispensing 6.5 microliters of transfection mix per well before culturing the cells for further 36 to 48 hours. To measure the BRET signal. First dissolve live cell luciferase substrate at 34 milligrams per milliliter in DMSO by vortexing.
Dilute reconstituted live cell luciferase substrate at one to 1000 in substrate dilution medium prewarm 37 degrees Celsius. Allow 50 microliters of substrate dilution medium per well vortex to mix. A precipitate may form, but will not interfere with the assay.
Aspirate the culture medium from the 96 well plate dispense 50 microliters of diluted live cell luciferase substrate into each well after culturing the cells for at least two hours. Remove the lid from the 96 well plate and incubate the plate for 10 minutes at room temperature inside the luminometer. Measure emission from LUCIFERASE and YFP one well at a time according to the details in the text protocol, integrate emission signals over 10 seconds.
Members of the Fox P family of transcriptional Repressors play a role in brain development. Mutations have been implicated in speech and autism spectrum disorders. FOX P two is known to form homodimers to validate the Brett assay for the detection of Fox.
P two Homodimers cells were transfected with constructs for expression of luciferase as the donor or YFP as the acceptor fused to either Fox P two or a nuclear localization signal. The nuclear targeted LUCIFERASE and YFP proteins serve as negative controls as a positive control for detection of a Brett. Signal cells were also transfected with a construct for expression of a luciferase YFP fusion protein.
The increase in the BRET signal shows that the Bret assay is effective in detecting Fox P two homodimers fluorescence microscopy images of cells transfected with YFP fused to a nuclear localization signal, or with YFP fused to FOX P two are shown here. The specificity of the interaction was demonstrated by introducing a mutation into FOX P two, which is known to disrupt dimerization. When luciferase Fox P two delta E 400 and YFP Fox B two fusion proteins were co-expressed.
A reduction in the breath signal was observed compared to when Fox B two and YFP Fox B two were co-expressed. This signal reduction was not due to an alteration in the subcellular localization of the mutant protein to assess the effectiveness of the Bret assay in detecting typic interactions, the interaction of Fox P two with Fox P one was tested, A Brett signal was observed in both possible configurations of the assay. In addition, the suitability of the Brett assay or detecting interaction of Fox P two with non FOX P proteins was tested by examining the interaction with CT BP one, a transcriptional co repressor and known Fox P two interaction partner.
The Fox P two CT BP one interaction was detected by the Brett assay when Fox P two was the donor fusion protein and CT P one was the acceptor, but not in the reverse configuration. The interaction between FOX P two and CT P one was observed even though only a minor proportion of CT BP one was localized to the nucleus highlighting the sensitivity of this assay. Finally, the Bret assay was employed to examine the effects of pathogenic mutations in Fox P two on protein protein interactions.
Two point mutations in Fox P two have been reported to cause a rare autosomal dominant disorder affecting speech and language. The ability of the mutant proteins to dimerize with wild type fox P two was assessed using the BRET assay. The R 3 28 X, but not the R 5 53 H mutation, disrupts dimerization the results using wild type Fox B two as a donor and mutant Fox B two.
As the accepter are shown, the same results were observed for the reverse configuration. After watching this video, you should have a good understanding of how to apply the bread assay to monitor protein protein interactions. This technique paved the way for researchers in the field of cell biology to explore protein-protein interactions in live cells.
Following this procedure, additional techniques can be performed to assay the activity of your protein of interest in order to assess the functional consequences of an observed protein protein interaction.
