Yeast cultures were grown in YPD media at 30°C to mid-log phase and extracts were prepared as previously described22 (link),23 (link). Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane. Anti-H3K9ac (Millipore, 07-352) and anti-H3K9cr (PTM Biolabs, PTM-516) were diluted to 1:2000 and 1:1000, respectively, in 1x Superblock (ThermoScientific). An HRP-conjugated anti-rabbit (GE Healthcare) was used for detection. Bands were quantified using the ImageJ program.
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Histone H3 Acetylation and Crotonylation Assay
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Corresponding Organization :
Other organizations : University of Colorado Denver, University of North Carolina at Chapel Hill, The University of Texas MD Anderson Cancer Center
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Variable analysis
independent variables
- Yeast culture growth in YPD media
- Protein expression and post-translational modifications (H3K9ac and H3K9cr)
- Growth temperature (30°C)
- Growth to mid-log phase
- Cell lysis and protein extraction method
- SDS-PAGE and PVDF membrane transfer
- Antibody concentrations (1:2000 for anti-H3K9ac, 1:1000 for anti-H3K9cr)
- HRP-conjugated anti-rabbit for detection
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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