Our efforts are currently focused on better characterizing the existing correlation between epigenetic changes and metabolic rewiring, with the ultimate goal of identifying novel epigenetic therapeutic targets. Given the dynamic and the reversible nature of epigenetic modification, recently, several epi-drug agents have been developed, leading to the improvement of cancer treatment and the reduction of chemo resistance. Recently, significant progress has been made in the field of instance characterizations, thanks to the application of modern mass spectometry, coupled with the separation techniques, such as top page or high-performance liquid chromatography.
The main challenge in histone characterization is, the existence of uncountable histone proteoforms, resulting from the crosstalk between dynamic modified enzyme and metabolites'availability. Begin by preparing a mini TAU gel for a mini vertical polyacrylamide gel, electrophoresis system. To do so, dissolve six molar urea in a solution of 15%acrylamide bis-acrylamide, and one milliliter of ultrapure water under gentle agitation at room temperature.
Then add the other components and adjust the volume. Fill the preset gel plates with this solution, ensuring no air bubbles form, and leaving 1.5 centimeters from the top. Add one milliliter of isopropanol on top to level the gel.
To prepare the stacking gel dissolve, dissolve urea in a 6%acrylamide, bis-acrylamide with one milliliter of ultrapure water. After adding the other components, fill the gel plates with this solution and insert a 10 well comb between the plates until the solution reaches halfway up the comb's teeth. Next dissolve six molar urea, 5%glacial acetic acid, and double distilled water to prepare 100 microliters of TAU sample buffer.
Then dissolve the lyophilized, histone sample in 30 microliters of TAU sample buffer. Fill the mini cassette with TAU running buffer with a final concentration of 5%acetic acid. Use a syringe to rinse the gel's wells with running buffer, and load the sample into the wells with a micro pipette.
Then connect the electrophoresis chamber to the power supply, placing the positive electrode at the bottom. Occasionally, turn off the power supply and rinse the gel wells with a syringe to remove bubbles from the gel's bottom surface. After removing the gel from the gel plates, cut the lane of interest, using a clean scalpel.
Now wash the gel lane with 15 milliliters of 0.5 M tris hydrochloride, pH 8.8 for 15 minutes. Prepare 15 milliliters of equilibration buffer one and two, using the components shown here. Then store equilibration buffer two in the dark.
Now, incubate the gel lane in 10 milliliters of equilibration buffer one with gentle agitation for 15 minutes, then decant. After that, incubate the gel lane in 10 milliliters of equilibration buffer two under gentle agitation for 15 minutes in the dark before decanting it. Next, prepare a 15%acrylamide resolving gel, using the composition shown here.
Set up the gel plate and fill it with the resolving gel solution, leaving 1.5 centimeters from the end of the long gel plate. Add one milliliter of isopropanol on top to level the gel and allow it to polymerize. Once the gel has polymerized, decant the isopropanol and dry the well, using three mm cellulose chromatography paper.
Insert the cut gel lane into the resolving gel well, using tweezers to prevent air bubbles, then seal the gel lane to the resolving gel, using 0.5%low melting point agarose in tris glycine and SDS buffer, with five microliters of 0.003%bromophenol blue. Place the gel plates in the cassette and fill them with tris glycine and SDS buffer. Run the gel at 120 volts until the bromophenol blue dye reaches the bottom.
After running, gently remove the gel from the plates and discard the excess agarose. To begin, perform mini 2D gel triton acid urea, or TAU electrophoresis on histone samples, followed by silver staining. Then prepare the reagents shown here.
Using a clean scalpel, excise histone spots of interest from the stained gel, and place each spot into a fresh, 1.5 milliliter silicon tube to avoid isoform cross-contamination. Add 250 microliters of 50 millimolar ammonium bicarbonate, or 50%acetone nitro to each gel spot and incubate under gentle agitation in a thermo mixer at room temperature. Decant the solution and repeat this process three times until the gel pieces appear transparent.
To dehydrate the gel spots, add 200 microliters of acetone nitrol. During this step, the gel pieces will turn opaque white. Decant the acetone nitrile, and allow the gel spots to air dry for 10 minutes.
The gel pieces will appear dry in the tubes. Next, rehydrate the gel spots by adding 20 microliters of trypsin solution or enough volume to cover the gel pieces. Incubate the tubes in a thermo mixer at 37 degrees Celsius under gentle agitation for at least four hours and up to 16 hours.
Transfer the supernatant, containing the triptych peptides to a fresh tube. Add 50 microliters of extraction solution to the gel pieces and sonicate twice in an ultrasonic water bath for 10 minutes at four degrees Celsius. Transfer the supernatant, containing the additional triptych peptides to the same fresh tube.
Use a lyophilizer to dry the pooled extracted peptides at 30 degrees Celsius. A lyophilized pellet will form in the tube. Now, add 15 microliters of 0.1%trifluoroacetic acid to each tube, and gently incubate in a thermo mixer at 25 degrees Celsius.
Transfer the supernatant to a vial for liquid chromatography tandem, mass spectrometry analysis.
