As the quality and specificity of antibodies is an ongoing issue in biomedical research1, 2, there has been a growing demand for reliable controls. EpiCypher’s SNAP-ChIP® approach is rapidly becoming the new standard for controlling chromatin immunoprecipitation (ChIP) experiments to histone post-translational modifications (PTMs)3.
What is SNAP-ChIP?
SNAP-ChIP (Sample Normalization and Antibody Profiling Chromatin ImmunoPrecipitation) uses DNA-barcoded, recombinant designer nucleosomes (dNucs) as spike-in controls to monitor antibody binding specificity and enrichment in ChIP experiments3, 4. Importantly, SNAP-ChIP can be easily incorporated into existing ChIP workflows, allowing unparalleled control in monitoring ChIP experiments.
We encourage the use of SNAP-ChIP spike-ins in every ChIP, as they empower scientists to monitor experimental variation, avoid sequencing poorly prepared samples, provide validation during peer review, and enable accurate normalization across samples3, 4. Thus, SNAP-ChIP spike-in controls increase confidence in experimental results and assurance that biological findings can be specifically attributed to the target of interest.
Introducing the K-AcylStat
SNAP-ChIP Spike-In Panel
Last week, EpiCypher launched an exciting new line of products in the SNAP-ChIP family : K-AcylStat™ SNAP-ChIP spike-in panel and K-AcylStat™ SNAP-ChIP certified antibodies. This advancement expands the application of SNAP-ChIP technology to diverse lysine acylation (i.e. K-Acyl) PTMs, including disease-relevant histone acetylation, as well as extended acylation states with emerging importance in epigenetic biology, such as histone butyrylation and crotonylation5 (Figure 1).
The K-AcylStat Spike-In Panel
Enables the Study of Combinatorial PTMs
The K-AcylStat SNAP-ChIP panel includes 22 distinct modified nucleosomes, plus an unmodified control (Figure 1). Notably, this set contains four nucleosomes harboring combinatorial PTMs, including the widely studied H3K27ac PTM in combination with nearby phosphorylation at S28 (H3K27ac + S28ph)6, 7, as well as tetra-acetylated H3, H4, and H2A.
The impact of physiological co-occurring PTMs on chromatin is gaining recognition in the field, and has been shown to impact antibody binding on chromatin 6-16. Indeed, there have been multiple studies describing the polyacetylated chromatin preference of site-specific H4 K-acetyl antibodies 7, 9. In contrast, many PTM epitopes are masked or altered in the presence of adjacent PTMs, as is the case with H3K27ac and H3S28ph6, 7.
Antibody Binding can be Altered
by Neighboring PTMs
As part of EpiCypher’s research program, the new K-AcylStat panel was used to screen >100 commercially available ChIP-grade antibodies to various lysine acylation PTMs. These experiments demonstrate that many acetylation-specific antibodies are impacted by adjacent modifications. For instance, in testing H3K18ac antibodies, some antibodies had equal binding on poly-acetylated vs. single-acetyl nucleosome substrates (Figure 2A), while other antibodies clearly preferred a poly-acetylated nucleosome (Figure 2B). This distinction is important to know when analyzing your ChIP-seq results, as differential binding to various acetyl forms could greatly impact (or complicate) interpretations.
Acetyl-Specific Antibodies can
Cross-React with Exotic PTMs
Another interesting trend EpiCypher observed is that many acetyl-specific antibodies also recognize the structurally related butyryl and crotonyl marks. This phenomenon was particularly evident in testing H3K9ac antibodies, which revealed different affinities of H3K9ac antibodies for the butyryl PTM (Figure 3A vs 3B). As extended acyl states are rapidly gaining appreciation for their important roles in epigenetic biology and disease5, 17-19, the ability of antibodies to distinguish between these PTMs is critical for deciphering experimental results.
K-AcylStat SNAP-ChIP Certified Antibodies :
Better Reagents for a Better ChIP
Along with the launch of the K-AcylStat SNAP-ChIP spike-in panel, EpiCypher is also releasing a complementary set of SNAP-ChIP certified antibodies. Thus, among >100 antibodies tested, the highest quality reagents exhibiting superior specificity and efficiency of target recovery are now widely available!
Although EpiCypher has done the hard work of identifying these gold-standard reagents among the myriad commercial “ChIP-grade” antibodies, the inclusion of SNAP-ChIP as a spike-in control for every experiment is strongly recommended (click here for more info).
The list of SNAP-ChIP Certified Antibodies is rapidly growing to fill demands in the field; check this page for the most up-to-date collection of SNAP-ChIP validated antibodies. In addition, EpiCypher has recently announced a partnership with ThermoFisher Scientific® to produce best-in-class PTM antibodies. The K-AcylStat panel is an essential step toward the generation of highly specific antibodies and related tools for the chromatin research. Stay tuned for updates; it’s going to be an exciting year!
1. Baker M. Reproducibility crisis: Blame it on the antibodies. Nature, 2015. 521(7552): p. 274-6. (PubMed PMID: 25993940)
2. Bradbury A, Pluckthun A. Reproducibility: Standardize antibodies used in research. Nature, 2015. 518(7537): p. 27-9. (PubMed PMID: 25652980)
3. Shah RN, et al. Examining the Roles of H3K4 Methylation States with Systematically Characterized Antibodies. Mol Cell, 2018. 72(1): p. 162-77 e7. (PubMed PMID: 30244833) (PMC6173622)
4. Grzybowski AT, et al. Calibrating ChIP-Seq with Nucleosomal Internal Standards to Measure Histone Modification Density Genome Wide. Mol Cell, 2015. 58(5): p. 886-99. (PubMed PMID: 26004229) (PMC4458216)
5. Tan M, et al. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell, 2011. 146(6): p. 1016-28. (PubMed PMID: 21925322) (PMC3176443)
6. Lau PN, Cheung P. Histone code pathway involving H3 S28 phosphorylation and K27 acetylation activates transcription and antagonizes polycomb silencing. Proc Natl Acad Sci U S A, 2011. 108(7): p. 2801-6. (PubMed PMID: 21282660) (PMC3041124)
7. Bock I, et al. Detailed specificity analysis of antibodies binding to modified histone tails with peptide arrays. Epigenetics, 2011. 6(2): p. 256-63. (PubMed PMID: 20962581) (PMC3230550)
8. Fuchs SM, et al. Influence of combinatorial histone modifications on antibody and effector protein recognition. Curr Biol, 2011. 21(1): p. 53-8. (PubMed PMID: 21167713) (PMC3019281)
9. Rothbart SB, et al. Poly-acetylated chromatin signatures are preferred epitopes for site-specific histone H4 acetyl antibodies. Sci Rep, 2012. 2: p. 489. (PubMed PMID: 22761995) (PMC3388470)
10. Duan Q, et al. Phosphorylation of H3S10 blocks the access of H3K9 by specific antibodies and histone methyltransferase. Implication in regulating chromatin dynamics and epigenetic inheritance during mitosis. J Biol Chem, 2008. 283(48): p. 33585-90. (PubMed PMID: 18835819) (PMC2586264)
11. Fuchs SM, Strahl BD. Antibody recognition of histone post-translational modifications: emerging issues and future prospects. Epigenomics, 2011. 3(3): p. 247-9. (PubMed PMID: 22122332)
12. Hirota T, et al. Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin. Nature, 2005. 438(7071): p. 1176-80. (PubMed PMID: 16222244)
13. Morrison EA, et al. The conformation of the histone H3 tail inhibits association of the BPTF PHD finger with the nucleosome. Elife, 2018. 7: p. (PubMed PMID: 29648537) (PMC5953545)
14. Gatchalian J, et al. Accessibility of the histone H3 tail in the nucleosome for binding of paired readers. Nat Commun, 2017. 8(1): p. 1489. (PubMed PMID: 29138400) (PMC5686127)
15. Morrison EA, et al. DNA binding drives the association of BRG1/hBRM bromodomains with nucleosomes. Nat Commun, 2017. 8: p. 16080. (PubMed PMID: 28706277) (PMC5519978)
16. Weaver TM, et al. Reading More than Histones: The Prevalence of Nucleic Acid Binding among Reader Domains. Molecules, 2018. 23(10): p. (PubMed PMID: 30322003) (PMC6222470)
17. Zhao D, et al. YEATS Domain-A Histone Acylation Reader in Health and Disease. J Mol Biol, 2017. 429(13): p. 1994-2002. (PubMed PMID: 28300602)
18. Rousseaux S, Khochbin S. Histone Acylation beyond Acetylation: Terra Incognita in Chromatin Biology. Cell J, 2015. 17(1): p. 1-6. (PubMed PMID: 25870829) (PMC4393657)
19. Dutta A, et al. Diverse Activities of Histone Acylations Connect Metabolism to Chromatin Function. Mol Cell, 2016. 63(4): p. 547-52. (PubMed PMID: 27540855) (PMC5298895)