Epigenetics is the study of heritable phenotypic changes that alter gene expressions but do not alter the DNA sequence. Every cell in the human body has the same DNA; epigenetics determines whether a cell becomes a neuron or a liver cell, for example. This differentiation is determined by what genes have been activated and what genes have been silenced. Epigenetics also plays a role in DNA compaction and genomic stability. Epigenetic antibodies are a common reagent used for epigenetic studies in western blots, ChIP, and immunofluorescence.
Much of epigenetics is controlled by post-translation modifications (PTMs) on DNA, RNA, and proteins. PTMs such as methylation, acylation, phosphorylation, and ubiquitination modify the accessibility of the DNA or recruit proteins that silence or activate transcription. The modifications can remain during cell division and, in some cases, are inherited through generations. Many diseases are often linked to an epigenetic dysregulation where there is inappropriate activation or silencing. These diseases include many types of cancer, autoimmune disorders, and neurological disorders. Epigenetics plays a crucial role in gene regulation; many researchers rely on sensitive specific epigenetic antibodies for their experiments. Thermo Fisher Scientific offers Invitrogen products that include a comprehensive library of primary antibodies; a growing number of these epigenetic antibodies are recombinant.
Types of Epigenetic PTMs
Histone modifications serve as both activating and repressive marks and include methylation, acetylation, crotonylation, phosphorylation, and ubiquitination. Histones are the protein components that form nucleosomes to compact the DNA into the basic units of chromatin. Specificity is of the utmost importance for histone antibodies because an antibody needs to be able to distinguish between very small differences such as a di- or tri-methylation.
Histone modifications are regulated by writers, readers, and erasers.
- Writers are the enzymes that place the mark on a histone such as histone acetyltransferase (HAT) or histone methyltransferase (HMT).
- Readers bind to the epigenetic mark and include bromodomain, chromodomain, and Tudor proteins.
- Erasers are the enzymes that remove the marks such as a histone deacetylase (HDAC) and lysine demethylase (KDM).
DNA methylation has classically been associated with gene silencing where methylation occurs primarily on the cytosine (C) of CpG dinucleotides where a DMNT enzyme converts the C to a 5mC (5-methylcytosine). DNA methylation is considered a rather stable modification compared to other epigenetic modifications. However, research over the course of the last decade has revealed that DNA methylation is much more dynamic and that there is a cycle of DNA methylation and demethylation which requires TET enzymes. DNA methylation is found beyond CpG islands and at transcriptional start sites, in gene bodies, and regulatory and repeat sequences. The dynamics and context of DNA methylation reveals new avenues for researchers to examine.
Our growing portfolio of traditional and recombinant antibodies is designed to enable detection and characterization of epigenetic targets with exceptional specificity to particular post-translational modifications and reproducibility in the form of antibody lot-to-lot consistency.
Immunofluorescence staining of antibody specificity for phosphorylated proteins.Phospho-Histone H2A.X (Ser139) Monoclonal Antibody (3F2), Cat. No. MA1-2022, was used for analysis following treatment with paraquat and iron which induces MnSOD and phosphorylation of H2AX in RAW 264.7 macrophages. Cells were treated for 20 hours with paraquat (500 µM) and iron (200 µg/mL) and stained with anti-Phospho-H2AX antibody.
Antibody specificity was demonstrated by siRNA-mediated knockdown of target protein. HeLa cells were transfected with SUV39H1 siRNA and a decrease in signal intensity was observed when compared to controls by western blot analysis (panel a) using SUV39H1 Recombinant Rabbit Monoclonal Antibody (3H60L17), Cat. No. 702443. Densitometric analysis of the western blot (panel b) quantifies loss of signal in siRNA-mediated knockdown confirming antibody-specificity.
Antibody specificity was demonstrated by detection of enrichment of the target protein at specific gene loci. Chromatin Immunoprecipitation (ChIP) was performed using EZH2 Monoclonal Antibody (144CT18.104.22.168), Cat. No. MA5-18108 and PCR primer pairs that bind to active loci in the promoters of HOXA9, CCND2 (positive) and inactive loci of SAT2 and SATa satellite repeats (negative).
Antibody specificity was demonstrated by CRISPR-Cas9 mediated knockout of target protein. Loss of signal was observed for target protein in DNMT1 knockout (KO) 293T cell line using DNMT1 Polyclonal Antibody, Cat. No. PA5-30581.
Dot blot of double stranded DNA using 5-Methylcytosine Recombinant Rabbit Monoclonal Antibody, Cat. No. MA5-24694. The membrane was pre-spotted with 50, 5, and 0.5 ng/dot of double stranded 5-Hydroxymethylcytosine (5-hmC) DNA, 5-Methylcytosine (5-mC) DNA, and unmethylated DNA.