Epigenetics is the study of heritable changes in gene expression that modify DNA, RNA and protein but do not alter the primary sequence. Post-translational modifications (PTMs) are one of the most common methods for regulating epigenetic states. Many types of proteins are subject to PTM and one of the most highly decorated is histones.
Histones package genomic DNA into nucleosomes, and this allows the approximately 2 meters of DNA to fit into a cell’s nucleus. The nucleosomes contain two subunits, each made of histones H2A, H2B, H3, and H4. Additionally, there is histone H1, which is often called the linker histone. The most prevalent PTMs found on histones are methylation, acetylation, phosphorylation, and ubiquitination.
We offer specific antibodies to most histone modifications. We also have hundreds of antibodies to detect other epigenetic and transcription factors. A growing number of our antibodies for epigenetics research have been developed using Invitrogen™ ABfinity™ antibody technology to create recombinant rabbit monoclonal and oligoclonal antibodies. ABfinity antibodies are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance.
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Antibodies for specific & reproducible epigenetic target detection
Our growing portfolio of traditional and recombinant antibodies is designed to enable detection and characterization of epigenetics targets with exceptional specificity to particular post-translational modifications and reproducibility in the form of antibody lot-to-lot consistency.
No detectable variation in three lots of H3K9me3 antibody. Immunofluorescence was performed using three different lots of histone H3K9me3 ABfinity™ recombinant rabbit oligoclonal antibody (Cat. No. 710816, 0.5 mg/mL) on fixed and permeabilized HeLa cells for detection, followed by labeling with goat anti–rabbit IgG (H+L) Superclonal™ secondary antibody, Alexa Fluor™ 488 conjugate (Cat. No. A27034, 0.4 µg/mL, diluted 1:2,500). Left panels reveal nuclear localization of histone H3K9me3 protein (green) via the antibodies. Middle panels show nuclear staining (blue) by SlowFade™ Gold Antifade Mountant with DAPI (Cat. No. S36938, diluted 1:50). Right panels are composite images, plus cytoskeletal F-actin staining (red) using Alexa Fluor™ 594 Phalloidin (Cat. No. A12381, diluted 1:200). These data demonstrate excellent lot-to-lot consistency in detection of histone H3K9me3 by this ABfinity antibody.
H3K18ac is detected when cells are treated with histone deacetylase (HDAC) inhibitors. Western blot analysis of H3K18ac was performed on cells treated with sodium butyrate (5 mM for 24 hr) or SAHA (0.5 µM for 24 hr) as indicated at the bottom of the blot. Nuclear-enriched cell extracts from HeLa, HCT116, Jurkat, and K562 cells, treated as indicated, were prepared, and 30 µg of extract were run on a NuPAGE™ 4–12% Bis-Tris Gel (Cat. No. NP0321BOX) on an XCell SureLock™ Electrophoresis System (Cat. No. EI0002) with Novex™ Sharp Pre-Stained Protein Standard (Cat. No. LC5800). Resolved proteins were then transferred to a nitrocellulose membrane with the iBlot™ Dry Blotting System (Cat. No. IB21001). The blot was probed with anti–histone H3K18ac rabbit polyclonal antibody (Cat. No. 720095) and detected by chemiluminescence after using goat anti–rabbit IgG (H+L) Superclonal™ secondary antibody, HRP conjugate (Cat. No. A27036). A clear 17 kDa band corresponding to histone H3K18ac was observed across cell lines tested. The blot was reprobed with beta-tubulin antibody (Cat. No. 32-2600) as a loading control.
Post-translational modifications (PTMs) relevant to epigenetics research
- Methylation—one, two, or three methyl groups are added to a lysine or arginine. In the case of histones, histone methyltransferases (HMTs) have specificity for the histone residues and mono‑, di-, or trimethylation.
- Acetylation—the acetyl group from acetyl coenzyme A is added to specific histone lysine residues by histone acetyltransferases (HATs), and acetyl groups are removed by specific histone deacetylases (HDACs). Acetylation is generally associated with gene activation.
- Phosphorylation—kinases phosphorylate specific serines, threonines, or tyrosines on histones, and dephosphorylation is carried out by phosphatases. Phosphorylation often occurs during DNA repair and mitosis.
- Ubiquitination—ubiqutin is added by an E3 ligase and removed by a deubiquitinating enzyme (DUB). Although ubiquitination often is a mark for protein degradation, in this case ubiquitination is an epigenetic mark.
More about epigenetics and available antibodies
Methylation, acetylation, phosphorylation and ubitquitination marks on histones are key players in gene expression. These marks serve as signals for opening and compaction of the chromatin, as well as recruiting factors that promote and antagonize transcription.
Important sites of histone post-translational modification affecting epigenetics.
It is essential to use an antibody that is specific to an individual histone modification because each one represents a unique signal for gene expression. For example, Lys9 on H3 can be acetylated or methylated. Acetylation is an activating mark, and methylation has different signals depending upon the number of methyl groups. H3K9me1 is found enriched at transcription start sites, whereas H3K9me2 and H3K9me3 are associated with gene repression. Further, H3K9me2 is specifically associated with chromosome X inactivation. Thus each modification on H3K9 has a distinct effect on the cell and knowing the identity of the modification is essential for accurately characterizing expression.
H3K9me2 antibody specifically recognizes di-methyl at Lys9. Cross-reactivity ELISA for demonstrating specificity towards di-methyl-histone H3 Lys9 (Histone H3K9me2) was performed using histone H3K9me2 ABfinity™ recombinant rabbit monoclonal antibody (Cat. No. 701783). The antigens (H3K9me1, H3K9me2, H3K9me3, and H3K9 Nme) were coated at 0.004 µg/mL. Antibodies at 10, 2.5, 0.625, and 0.156 µg/mL were added, and the signal was detected using goat anti–rabbit IgG (H+L) secondary antibody, HRP conjugate (Cat. No. G-21234, diluted 1:5,000). The plate was developed using Stabilized Chromogen, TMB (Cat. No. SB02) and detected at an absorbance of 488 nm.
Antibodies against histone modifications must be specific and sensitive. Using H3K4me1 as an example, the H3K4me1 antibody must only recognize a single methyl group on Lys4 of H3 and not di- or trimethylation. Our ChIP-validated H3K4me1 antibody has greater specificity for that single modification and yields overall higher signal compared to two competitor antibodies tested in a peptide array (see figure). Specificity of this antibody has also been tested in a biological setting. SETD7 mono-methylates H3 at Lys4. PFI-2 is a potent inhibitor of SETD7. Using this antibody we were able to detect a decrease in H3K4me1 in nuclear lysates of cells treated with PFI-2 (see figure). This recombinant oligoclonal antibody has been shown to work in numerous applications: WB, flow cytometry, IHC, IF, and ChIP.
Histone H3K4me1 detection with superior peptide specificity. Peptide arrays were performed using our ABfinity™ recombinant rabbit oligoclonal antibody (Cat. No. 710795) and two other suppliers’ antibodies targeting H3K4me1. Arrays were incubated overnight with a 1:2,000 dilution of primary antibody. After washing, they were incubated with goat anti–rabbit IgG (H+L) Superclonal™ secondary antibody (Cat. No. A27036) at a dilution of 1:5,000 for 1 hr. Arrays were incubated with SuperSignal™ West Pico substrate (Cat. No. 34078) and then visualized using the myECL™ Imager (Cat. No. 62236).
Identification of a decrease in H3K4me1 in PFI-2 treated cells using H3K4me1 oligoclonal antibody, Cat. No. 710795. HeLa cells were treated with varying concentrations of SETD7 inhibitors as indicated for 2 hr. Cells were collected, and chromatin-bound nuclear extracts were prepared. Western blot analysis of H3K4me1 (upper panel) was performed by loading 10 µg of acid chromatin-bound nuclear extract in reducing sample buffer (Cat. No. 39000) and protein ladder (Cat. No. 26619) onto a 4–20% Tris-glycine polyacrylamide gel (Cat. No. WT4201BX10). Proteins were transferred to nitrocellulose membrane (Cat. No. 88018) with Pierce™ 1-Step Transfer Buffer (Cat. No. 84731) using a semi-dry blotter (Cat. No. 62288). Membrane was blocked in StartingBlock™ T20 (Cat. No. 37543) for 30 min at room temperature.
(Figure caption continued) H3K4me1 was detected at approximately 17 kDa using a histone H3K4me1 ABfinity™ recombinant rabbit oligoclonal antibody (Cat. No. 710795) at a dilution of 1:2,000 in StartingBlock T20 overnight at 4°C on a rocking platform, followed by a goat anti–rabbit Superclonal™ IgG-HRP secondary antibody (Cat. No. A27036) at a dilution of 1:5,000 for 1 hr. Chemiluminescent detection was performed using SuperSignal™ West Pico substrate (Cat. No. 34078) and the myECL Imager (Cat. No. 62236). A loading control (lower panel) was performed by stripping the blot with Restore™ PLUS Western Blot Stripping Buffer (Cat. No. 46430) and reprobing with histone H3 ABfinity™ oligoclonal antibody (Cat. No. 711055) at a dilution of 1:2,000 in StartingBlock T20 overnight at 4°C on a rocking platform, followed by goat anti–rabbit Superclonal™ IgG-HRP secondary antibody (Cat. No. A27036) at a dilution of 1:5,000 for 1 hr.
Antibodies targeting common histone modifications
Epigenetic regulation is dynamic and includes writers, erasers, and readers. Writers place a mark on a specific amino acid on histones or other proteins. These include histone acetyltransferases (HATs), histone methyltransferases (HMTs), protein arginine methyltransferases (PRMTs), and kinases. Erasers remove such marks and include histone deacetylases (HDACs), lysine demethylases (KDMs), and phosphatases. Readers bind to the epigenetic marks and include proteins with bromodomains, chromodomains, and Tudor domains. The writing, reading, and erasing of these posttranslational marks lead to changes in chromatin structure that can promote or antagonize gene expression. This highly dynamic process regulates transcription, DNA replication, and DNA repair. Mutations in many of these proteins are associated with disease.
We offer antibodies to detect most of these writer, reader, and eraser targets for a variety of applications: western blot, IF, IHC, IP, flow cytometry, and ChIP.
Writers, readers, and erasers dynamically associate with chromatin and regulate gene expression. Reprinted by permission of MacMillan Publishers Ltd: Nature Reviews Drug Discovery 13, 673–691 (2014). Visit http://www.nature.com/nrd/index.html.
Immunohistochemistry analysis of CBX5. showing staining in the nucleus of paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0) and heating by microwave for 8–15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a CBX5 monoclonal antibody (Cat. No. 730019), diluted in 3% BSA-PBS at a dilution of 1:100, overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST, and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.
Immunofluorescence analysis of PRMT5 performed on 70% confluent log-phase MCF-7 cells. The cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton™ X-100 for 10 min, and blocked with 1% BSA for 1 hr at room temperature. The cells were labeled with PRMT5 mouse monoclonal antibody (Cat. No. 730054) at 2 µg/mL in 0.1% BSA and incubated for 3 hr at room temperature and then labeled with goat anti–mouse IgG (H+L) Superclonal™ secondary antibody, Alexa Fluor™ 488 conjugate (Cat. No. A28175) at a dilution of 1:2,000 for 45 min at room temperature (green, Panel A). Nuclei (blue, Panel B) were stained with SlowFade™ Gold Antifade Mountant with DAPI (Cat. No. S36938). F-actin (red, Panel C) was stained with Alexa Fluor™ 555 Rhodamine Phalloidin (Cat. No. R415, diluted 1:300). Panel D is a merged image showing cytoplasmic localization. Panel E is a control with no primary antibody. The images were captured at 60X magnification.
Chromatin immunoprecipitation analysis of HDAC3. The experiment was performed using crosslinked chromatin from 1 x 106 HCT116 colon carcinoma cells treated with serum for 0, 15, and 30 min. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 µL per 100 µL well volume of an HDAC3 polyclonal antibody (Cat. No. PA1-862). Chromatin aliquots from ~1 x 105 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers to amplify 15 kb upstream of the Egr1 gene, or exon 1 or exon 2 of Egr1. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean ± SEM for three experiments. A schematic representation of the Egr-1 locus is shown above the data, where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions), the zigzag line represents an intron, and the straight line represents upstream sequence. Regions amplified by Egr-1 primers are represented by black bars. Data courtesy of the Antibody Data Exchange Program.
Table is sorted by histone (column 1), then by amino acid position of site (column 2). To find antibodies for writers, erasers, or readers of interest, search for the target using the antibody search tool at the top of this page.
|H2A||K5||ac||Tip60, p300, CBP, KAT1, KAT5|
|H2A||T120||ph||Bub1, VprBP, NHK-1|
|H2A.X||S139||ph||ATM, ATR, DNA-PK||PP4||MDC1, MDC1, NBS1, 53BP1, TDRr, BRCA1|
|H2B||K12||ac||p300, CBP, ATF2|
|H2B||K15||ac||p300, CBP, ATF2|
|H2B||K120||ub||UBE2E1, RNF20, RNF40, UBE2A, UBE2B|
|H3||K4||ac||SIRT1, SIRT2, SIRT3, HDAC1, HDAC2, HDAC3|
|H3||K4||me||MLL1, MLL2, MLL3, MLL4, MLL5, SETD1A, SETD1B, ASH1, SETD7, NSD3||LSD1, LSD2, KDM2B, JARID1A, JARID1B, JARID1C, JARID1D, PHF8, NO66||CHD1, MRG15, PHF20L1, TAF3, ING1, ING2, ING3, ING4, ING5, BPTF, RAG2, ATRX|
|H3||K9||ac||GCN5, PCAF, ELP3||SIRT6, SIRT1||BRD4, BAZ1B|
|H3||K9||me||Suv39H1, SUV39H2, G9a, SETDB1, Ash1, KMT1D, CLL8, RIZ1||LSD1, KMD3A, KMD3B, KMD4A, KMD4B, KMD4C, KMD4D, TRIP8, PHF8||L3MBTL1, Tip60, SFMBT, HP1, CDY1, PC1, MPP8, CBX1, CBX2, CBX3, CBX4, CBX5, CBX6, CBX7, CBX8, Np95, JARID1C, ATRX|
|H3||S10||ph||Aurora-B, MSK1, IKK-alpha, Snf1, MSK2, Pim1||PPF||14-3-3|
|H3||K14||ac||GCN5, PCAF, CBP, p300, Tip60, SRC-1, Elp3, KAT12, TAF1, MOZ, MORF||BRD4, BAZ1B, BRG1|
|H3||K18||ac||GCN5, p300, CBP, PCAF, KAT12|
|H3||K23||ac||GCN5, Sas3, p300, CBP, KAT3A, KAT3B|
|H3||K27||ac||GCN5, p300, CBP|
|H3||K27||me||EZH2, G9a, EZH1, NSD3||UTX, JMJD3, PHF8||PC1, CBX2, CBX4, CBX6, CBX7, CBX8, EED|
|H3||S28||ph||Aurora-B, MSK1, MSK2||14-3-3|
|H3||K36||me||SETD2, NSD1, SMYD2, NSD2, ASH1, SETMAR||KDM2A, KDM2B, KDM4A, KDM4B, KDM4C, NO66||MSL3, MRG15, BRPF1, PHF19, PHF1|
|H3||K56||ac||GCN5, CBP, p300||HDAC1, HDAC2, SIRT2, SIRT6|
|H4||R3||me||PRMT 1, PRMT5, PRMT6||JMJD6||TDRD3|
|H4||K5||ac||Hat1, Tip60, ATF2, p300, CBP, HBO1||BRD4|
|H4||K8||ac||GCN5, Tip60, ATF2, Elp3, p300, CBP, HBO1||BRD2, BRD4|
|H4||K12||ac||Hat1, Tip60, p300, CBP||BRD2, BRD4|
|H4||K16||ac||Gcn15, MOF, Tip60, ATF2||SIRT2, SIRT1|
|H4||K20||me||PR-SET7, SUV4-20H1, SUV20-H2, ASH1, NSD1, SETD8||PHF20L1, L3MBTL1, SFMBT, MBTD1, 53BP1|
Polycomb group (PcG) proteins function in polycomb repressive complexes (PRC1 and PRC2). PRC complexes modify histones as well as other proteins, and they are generally associated with silencing of gene expression. These complexes play key roles not only in epigenetic regulation of transcription but also in stem cell identity, differentiation, and disease. PRC1 and PRC2 interactions are dynamic: PRC2 is recruited to the chromatin and one of its components, EZH2, deposits the repressive trimethyl Lys27 on H3. CBX proteins, which form a component of PRC1, are recruited to the H3K27me3 mark. The RING proteins in PRC1 can act as an E3 ubiquitin ligase and ubiquitinate Lys119 on H2A—a critical silencing mark during development.
Functions of polycomb repressive complexes (PRC1 and PRC2).
Enrichment of endogenous histone H2A-Ub protein using anti–histone H2A-Ub rabbit polyclonal antibody. Chromatin immunoprecipitation (ChIP) was performed using anti–histone H2A-Ub rabbit polyclonal antibody (Cat. No. 720148, 3 µg) on sheared chromatin from 2 x 106 HeLa cells using the MAGnify™ Chromatin Immunoprecipitation System (Cat. No. 49-2024). Normal rabbit IgG (1 µg) was used as a negative IP control. The purified DNA was analyzed on the Applied Biosystems™ 7500 Fast Real-Time PCR System (Cat. No. 4351106) with optimized PCR primer pairs for the region of the inactive SAT2 satellite repeat used as the positive-control target, and promoters of the active cFOS (FOS) beta-actin (ACTB) region used as the negative-control target. Results are presented as fold enrichment of the antibody signal compared to the negative control IgG, using the comparative Ct method.
Flow cytometry analysis of EZH2 on HCT 116 cells. Cells were fixed with 70% ethanol for 10 min, permeabilized with 0.25% Triton™ X-100 for 20 min, and blocked with 5% BSA for 30 min at room temperature. Cells were labeled with EZH2 rabbit polyclonal antibody (Cat. No. 36-6300, red histogram) or with rabbit isotype control (pink histogram) at 3–5 µg per 106 cells in 2.5% BSA. After incubation at room temperature for 2 hr, the cells were labeled with goat anti-rabbit secondary antibody, Alexa Fluor™ 488 conjugate (Cat. No. A-11008) at a dilution of 1:400 for 30 min at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using the Applied Biosystems™ Attune™ Acoustic Focusing Cytometer. The purple histogram represents unstained control cells, and the green histogram represents a control with no primary antibody.
Transcription factors recognize and bind to specific sequences on DNA. They can activate or repress gene expression by altering RNA polymerase recruitment. Such factors can act alone or bind as a complex. Some factors bind unmodified proteins, while others require modification such as phosphorylation to bind. Some transcription factors are basal, while others respond to environmental or intracellular signals. Other factors are essential during development or differentiation. Transcription factors can also be involved in pathogenesis; transcription factors of this type include oncogenes and tumor suppressors.
Western blot analysis of NF-kappa-B (p65). The experiment was performed by running 20 µg of HeLa, A549, A431, K562, Jurkat, HEK-293, and MDA-MB-231 cell lysates on a NuPAGE™ 4–12 % Bis-Tris gel (Cat. No. NP0321BOX) in an XCell SureLock™ Electrophoresis System (Cat. No. EI0002) with the Novex™ Sharp Pre-stained Protein Standard (Cat. No. LC5800), and blotting the proteins using the iBlot™ Dry Blotting System (Cat. No. IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hr at room temperature. NF-kappa-B (p65) was detected at ~65 kDa using NF-kappa-B (p65) mouse oligoclonal antibody (Cat. No. 710048) at 0.5–1 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat anti–rabbit IgG HRP secondary antibody (Cat. No. G-21234) at 1:5,000 dilution was used, and chemiluminescent detection was performed using Novex™ ECL Chemiluminescent Substrate Reagent Kit (Cat. No. WP20005). The blot was reprobed with beta-tubulin (Cat. No. 32-2600) as a loading control.
ChIP-qPCR analysis of NF-kappa-B (p65) with specific antibody. The experiment was performed with 3 µg/mL of NF-kappa-B p65 ABfinity™ oligoclonal antibody (Cat. No. 710048) on sheared chromatin from 2 x 106 HeLa cells treated with TNF-alpha (50 ng/mL for 1 hr) using the MAGnify™ Chromatin Immunoprecipitation System (Cat. No. 49-2024). Normal rabbit IgG (3 µg/mL) was used as a negative IP control. The purified DNA from each ChIP sample was analyzed using the Applied Biosystems™ StepOnePlus™ Real-Time PCR System (Cat. No. 4376600), with primers for the promoters of the IL-8 and IL-6 genes used as positive-control targets, and the GAPDH gene used as negative-control target. Results are presented as fold enrichment of the antibody signal compared to the negative control IgG, using the comparative Ct method.
Expression of SMAD2 detected after transfection of HeLa cells with siRNA. Cells were transfected with 50 nM Silencer™ SMAD2 siRNA (Cat. No. 115714) (lane 3), transfected with 50 nM Silencer™ Negative Control siRNA (lane 2), or left untransfected (lane 1). Proteins from cells lysates were then separated by gel electrophoresis, transferred to membrane and detected by western blot using SMAD2 ABfinity™ monoclonal antibody (Cat. No. 700048, 0.5 µg/mL) and goat anti–rabbit IgG (H+L) Superclonal™ secondary antibody, HRP conjugate (Cat. No. A27036, 0.4 µg/mL, 1:2,500 dilution). The blot was reprobed with actin antibody (Cat. No. PA5-16914) as a loading control. The relative densities of the bands normalized to actin confirm silencing of SMAD2 expression and the specificity of the ABfinity™ antibody.
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