The overall goal of this experimental strategy is to investigate the functionality of disease associated non-coding variants. This method can help answer key questions in genomics, such as how genetic variants that don't actually change amino acid sequence, still contribute to disease phenotype. The main advantage of this technique is that is provides a streamlined process for assessing genetic variance for function activity and gives insight into regulatory proteins and pathways effected.
This technique can benefit the investigation of any disease with a candidate causal variant. Because the procedure for analyzing these variants is universal regardless of the phenotype being studied. Though this method can provide insight into human disease, it can also be applied to mutations in any organism.
Nuclear lysates will be prepared from B-lymphoblastoid cells in this experiment, however this procedure can be applied to most cell lines. After counting cells using a hemocytometer, spin down the desired number of cells for lysing. Aspirate the media, add 10 milliliters of ice-cold PBS, and spin down the cells again.
Wash the cells a second time with PBS, and remove the PBS after the spin. Resuspend the cell palate in ice-cold PBS using one milliliter of PBS per 10 to the seventh cells. Aliquot one milliliter of the cell suspension to 1.5 milliliter microcentrifuge tubes, so that each tube contains 10 to the seventh cells in PBS.
Centrifuge for two minutes and aspirate off the PBS. Add to a working stock of CE buffer, one millimolar dithiothreitol, or DTT, 1X phosphatase inhibitor, and 0.5 millimolar Phenylmethanesulfonyl fluoride, or PMSF. Resuspend each cell palette with 400 microliters of this buffer, and incubate on ice for 15 minutes.
Add 25 microliters of 10%Nonidet P-40 to each microcentrifuge tube, and mix by pipetting. Centrifuge at four degrees Celsius and maximum speed for three minutes. Decant and discard the supernatant.
Next, add to a working stock of NE-buffer, one millimolar DTT, 1X phosphoaase inhibitor, and one millimolar PMSF. Resuspend each cell palate with 30 microliters of this buffer, and mix by vortexing. Incubate at 4 degrees Celsius, in a tube rotator, for 10 minutes.
Centrifuge for two minutes. Collect the clear supernatant, which is the nuclear lysate. Aliquot the nuclear lysate before storing at minus 80 degrees Celsius to avoid multiple freeze thaw cycles that may degrade the protein.
Begin this procedure by preparing a 50 nanomolar working stock of the oligonucleotide, as described in the protocol text. Place the oligos in a heat block at 90 degrees Celsius for five minutes. After five minutes, turn off the heat block and allow the oligos to slowly cool down to room temperature for at least one hour prior to use.
Next prepare the electrophoretic mobility shift assay or EMSA gel. Remove the slide from a pre-cast, six percent tris-borate-EDTA, or TBE gel, and rinse under deionized water several times to remove any buffer from the wells. Assemble the gel electrophoresis apparatus, and check for leaks by filling the inner chamber with 0.5X TBE buffer.
If no buffer leaks into the outer chamber, fill the outer chamber, roughly, two thirds of the way. Pre-run the gel at 100 volts for 60 minutes. When the pre-run is complete, flush each well with 200 microliters of 0.5X TBE buffer.
Prepare a binding buffer master mix. In a microcentrifuge tube, mix these reagents that are common to all reactions. 10X binding buffer, DTT polysorbate, Poly(dI-dC)and salmon sperm DNA.
To set up the EMSA reactions, add nuclease-free water to each microcentrifuge tube, such that the final volume, following addition of all reagents, will be 20 microliters. Add the appropriate amount of master mix to each microcentrifuge tube. Add eight micrograms of nuclear lysate to the appropriate microcentrifuge tubes.
Include tubes containing the oligo, without nuclear extract, as negative controls. Add 50 femtomoles of oligo to the appropriate microcentrifuge tubes, and flick to mix. Briefly spin the contents to the bottom of the tube.
Incubate all tubes, for 20 minutes, at room temperature. Next, add two microliters of 10X orange loading dye to each microcentrifuge tube. Pipette up and down to mix.
Load the samples into the pre-run, six percent, TBE gel by pipetting up and down to mix, and then expelling each sample into a separate well. Run the gel at 80 volts for approximately 60 to 75 minutes, until the orange dye has migrated two thirds to three quarters of the way down the gel. When the run is complete, use a gel knife to pry the plastic cassette open.
Remove the gel from the cassette and place it in a container with 0.5 percent TBE buffer to keep it from drying out. Place the gel on the surface of an infrared and chemiluminescence imaging system. Eliminate any bubbles or contaminants that will disrupt the image.
Scan the gel using the scanning system software as described in the protocol text. To begin this procedure, prepare a five micromolar oligo working stock as described in the protocol text. Place the oligos in a heat block at 95 degrees Celsius for five minutes.
After five minutes, turn off the heat block and allow the oligos to slowly cool down to room temperature prior to use. Warm the following buffers to room temperature:binding buffer, low stringency wash buffer, high stringency wash buffer, and elution buffer. Prepare the binding mixtures for each variant.
Mix one volume of nuclear lysate with two volumes of binding buffer. Add 1X phosphatase inhibitor, 0.5 millimolar PMSF, and 1X binding enhancer. Add 10 microliters of five micromolar biotinylated capture DNA to each binding mixture.
Mix by flicking the tube several times. Incubate the binding mixtures for 20 minutes at room temperature. Next, add 100 microliters of streptavidin MicroBeads to each binding mixture.
Incubate for 10 minutes at room temperature. For each oligo probe being tested, place a binding column in the magnetic separator. Make sure the columns are labelled with the variant oligo that was used in the binding mixture.
Place a microcentrifuge tube directly under each binding column, and apply 100 microliters of binding buffer to rinse the column. Pipette the contents of each binding mixture into separate columns. Allow the liquid to flow completely through the column into the microcentrifuge tube.
Apply 100 microliters of low stringency wash buffer to the column and wait until the column reservoir is empty. Apply 100 microliters of low stringency wash buffer to the column again. Apply 100 microliters of high stringency wash buffer to the column, and wait until the column reservoir is empty.
Apply 100 microliters of high stringency wash buffer to the column again. Add 30 microliters of native elution buffer to the column and let stand for five minutes. Finally add an additional 50 microliters of native elution buffer to elute the bound transcription factors.
In order to explore the reproducibility of EMSA results, and the consequences of freeze thaw cycles, oligos containing the reference, or non-reference allele of a genetic variant were used to probe the same preparation of B-lymphocyte nuclear extract after multiple cycles of freezing and thawing. The blue arrow indicates the free probe. Binding of the transcription factors to the oligo is seen as a band in the top half of the gel, indicated by the red arrow.
These results indicate that freezing and thawing this particular nuclear extract up to five times has, seemingly, no effect on the integrity of the proteins. It is also important to optimize the signal to noise ratio for the EMSA. Various concentrations of oligos were used to probe a single preparation of nuclear lysate.
An increase in the intensity of the band, up to 100 femtomoles of oligo was observed. Representative results from a study of a lupus associated risk allele are shown here. The transcription factor, STAT 1, was first identified by DAPA followed by proteomic analysis.
A DAPA western blot then confirmed the identity of STAT 1, and the higher binding of the phosphorylated form of STAT 1 to the lupus risk oligo. Following this procedure, other techniques, such as mass spectrometry and western blot can be performed. This will help us to identify the regulatory protein showing allele dependent binding.
Reporter assays like luciferase can also be used to investigate changes in gene expression as a function of allele.
