Post-translational histone modifications panel (active motif)

The reason for doing the post-translational histone modifications panel was to quantify abundance of various histone marks in young and old midguts and to look for age-associated changes. We sent our samples (whole midguts flash frozen in liquid nitrogen, ~50 midguts per sample) to Active Motif (https://www.activemotif.com/), which is a company that performed the entire protocol and analysis. I've attached the protocol they performed on our samples specifically. Please contact the Active Motif for further support on the exact types of reagents, etc.

To answer your second question about alternatives to ChIP-seq, one could use CUT&RUN or CUT&TAG methods, which do not require as much input material.

ASSAY Protocol 

1.  Add beads to well in 25 ul assay buffer (1% BSA in  PBS containing 0.05 Tween-20) supplemented with protein, phosphatase and deacetylase inhibitors.  Beads containing the H3S10ph were miss-loaded and thus are absent from the experiment.

2. Add samples  to wells in 25 ul  assay buffer supplmented with inhibitors in duplicate as four-point 1.4 fold dilution series.

3.  Incubate 1 hour with agitation

4. Perform two 100ul washes with 1X Wash Buffer. Use plate magnet to collect beads. Keep on plate magnet throughout washes.

5. Add 50 ul  Biotinylated Histone H3 antibody diluted 1:125 in Assay Buffer AM3 for the high abundance PTM multplex assay.  Reporter dilution for the  low abundance PTM multiplex was increased from the standard 1:500 dilution to account for the dilute nature of the samples

6. Incubate 1 hour with agitation.

7. Wash as above

8. Add 50 ul of SAPE diluted 1:100 in Assay Buffer AM3.

9. Incubate 30 min with agitation.

10. Place plate on magnet and discard SA-PE. 

11. Remove plate from magnet. Resuspend beads in 100 ul 1X Wash Buffer.

12. Read assay on Luminex LX-200.

13.  Evaluate value data to identify wells with all beads in their respective responsive ranges

14. Perform H3-Total based normalization of PTM-bead MFIs.

Category