Detecting Histone Modifications in Yeast Cells with Neurodegenerative Protein Overexpression

Protocolo

1. Cell lysis and western blotting to detect histone post-translational modifications

1. Cell lysis
   1. Thaw yeast cell pellets on ice and resuspend cell pellets in 100 µL of dH₂O.
   2. To the cell resuspension, add 300 µL of 0.2 M NaOH and 20 µL of 2-mercaptoethanol. Resuspend by pipetting up and down.
   3. Incubate cells on ice for 10 min and then centrifuged at 3,200 x g on a tabletop centrifuge for 30 s at RT. Discard supernatant.
   4. Resuspend the cell pellet in 100 µL of 1x loading dye and boil for 10 min on a heating block.
      NOTE: The recipe for 6x loading dye can be found in the Table of Materials.
2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and membrane transfer
   1. Prepare the gel chamber by placing two gels in a gel holder filling the inner chamber to the top with running buffer and filling the outside chamber to the two-gel line mark.
   2. Load 15 µL of sample per well into a 10-well 12% polyacrylamide gel. For the protein ladder lane, load 5 µL.
   3. Run the gel for approximately 45 minutes at 150 V, or until the loading dye front reaches the bottom of the gel.
   4. While the gel is running, prepare for membrane transfer by soaking fiber pads (two per gel) in transfer buffer (Table of Materials) and soaking polyvinylidene fluoride (PVDF) membrane in methanol. Rinse the membrane in the transfer buffer before transfer.
      NOTE: The PVDF membrane should be cut to the size of the gel and use one PVDF membrane for every gel being transferred.
   5. Assemble, on a semi-dry transfer apparatus cell, a transfer ‘sandwich’: from the bottom electrode, place (1) a presoaked fiber pad, (2) presoaked and rinsed PVDF membrane, (3) gel, and (4) a second presoaked fiber pad.
      NOTE: To avoid air bubbles in the transfer ‘sandwich, ' gently roll the ‘sandwich’ with a serological pipet to force out bubbles. Once the gel has been placed on top of the PDVF membrane, do not move it.
   6. To carry out protein transfer, set the power pack to 150 mA for 1 h (for one ‘sandwich’).
3. Detection of histone post-translational modifications (PTMs) with modification-specific antibodies
   1. Remove the membrane from the transfer apparatus. Rinse briefly with dH₂O.
      1. Optionally, check the transfer of proteins with Ponceau-S stain by pouring enough Ponceau-S stain to cover the membrane in a small plastic box and incubating at RT with gentle shaking for 30−60 s. Remove the Ponceau-S stain and rinse with dH₂O until the background stain disappears and protein bands are visible to the naked eye. Continue to rinse with dH₂O until all stain is removed from the membrane.
   2. Block the membrane by incubating the blot for 1 h at RT with gentle rocking in tris buffered saline (TBS) blocking buffer (Table of Materials) in a small staining box. Use enough blocking buffer to cover the blot.
      NOTE: Be careful to place the membrane upright in the staining box so that the membrane side on which proteins lie is not facing down.
   3. Incubate blot in the staining box overnight with a histone modification-specific antibody reactive towards yeast at 4 °C. Dilute the antibody in TBS blocking buffer according to manufacturer specifications. Also include a proper nuclear loading control, such as anti-total H3 raised in a different host species from the modification-specific antibody. For instance, if probing for H3S10ph with an anti-H3S10ph antibody raised in rabbits, use an anti-total H3 antibody raised in mice as a loading control. Repeat this for every blot as necessary.
      NOTE: Antibody dilutions can be reused for a total of three times within a month. Store them at 4 °C.
   4. Wash the membrane 4x in house-made TBS + 0.1% polysorbate 20 (TBST) for 5 min with rocking at RT.
   5. Incubate blot with fluorescent secondary antibodies at manufacturer-specified dilutions (donkey anti-rabbit 680 and donkey anti-mouse) for 1 h at RT.
      NOTE: Fluorescent secondary antibodies must be protected from light during both storage and usage. Carry out incubation in dark plastic boxes or cover with aluminum foil.
   6. Wash membrane 4x with TBST for 5 min and wash with TBS for 5 min while rocking at RT.
   7. Visualize blot on a fluorescent Western blot imaging system for 2 min. Visualize the anti-rabbit 680 and anti-mouse 800 secondary antibodies in the 800 nm and 700 nm channels, respectively.
      NOTE: Replicates are independently conducted starting from section 1. For appropriate data interpretation in these experiments, it is necessary to verify that the antibody signal response is within the range over which the signal response is linear.

Materiales