Creative Biolabs provides premium antibodies designed to detect methylated proteins through our High-Affi™ platform. These reagents reliably identify endogenous concentrations of methylated lysine (mono-, di-, tri-methylated forms) and arginine residues (including mono- or asymmetrically/symmetrically di-methylated states). Notably, their specificity extends to distinct modification sites without reacting to acetylated proteins.

Background

Protein methylation dynamically modifies arginine or lysine residues post-translation. For arginine, peptidylarginine methyltransferases (PRMTs) drive either single methyl group addition (mono-methylation) or dual additions—either clustered on one nitrogen (asymmetric di-methylation) or split between two nitrogens (symmetric di-methylation). These modified arginines cluster in glycine-rich regions like RGG/RG motifs. Lysine residues accept up to three methyl groups via methyltransferases, while lysine demethylases (KDMs) strip these marks to maintain reversibility.

Fig.1 The process of lysine methylation and demethylation.^1,3

Though characterized in histones (where HMTs transfer methyl groups from SAM cofactors), methylation increasingly appears in non-histone proteins like p53 and estrogen receptor alpha. Both histone and non-histone methylation influence gene expression, protein production, and signaling cascades. This modification often partners with phosphorylation or acetylation to fine-tune protein behavior. Take p53: its monomethylation at K372 primes nearby lysines (K373/K382) for acetylation, boosting protein stability and tumor-suppressing activity. When methylation goes awry, consequences ripple through cell division, DNA repair, and survival pathways—mechanistically linking it to cancers, neurodegeneration, and heart disease.

Fig.2 Overview of histone methylation.^2,3

Antibody Types

Methylation-specific antibodies may be broadly categorized based on their specificity for many facets of protein methylation. Here are the main types that we provide, and these antibodies can be monoclonal, polyclonal, or recombinant.

Methylation Site-Specific Antibodies: These antibodies are made to identify methylation inside a given protein at a certain amino acid residue. An antibody could, for instance, bind especially to histone H3 exclusively in lysine 9 dimethylation (H3K9me2). These are absolutely essential for precisely determining the functional effects of methylation at specific sites.

Methyl-State Specific Antibodies: These antibodies distinguish among the many degrees of methylation on a given residue. This covers lysine antibodies, particularly for mono- (me1), di- (me2), or tri-methylated (me3). For arginine, this comprises antibodies either symmetric di-methylated (sdme2), asymmetric di-methylated (adme2), or mono-methylated (me1). These are fundamental for breaking apart the many functions of various methylation levels.

Pan Anti-Methyl Antibodies: These antibodies identify methylation residues independent of their particular place or protein of occurrence. Though less specific, they may be useful for enrichment or broad identification of methylation proteins.

Modification-Specific Antibodies (with Methylation Specificity): Some antibodies are designed to identify a particular methylation mark in concert with another alteration or a particular sequence context. An antibody may, for example, particularly identify a methylated lysine only when it is additionally phosphorylated or found in a certain amino acid sequence.

Discovery Strategy

Monospecific Anti-Methylation Polyclonal Antibody Production

Producing highly specific polyclonal antibodies targeting a defined methylation state requires immunizing animals, such as rabbits, with a synthetic peptide-carrier protein conjugate. This immunogen must incorporate the methylated residue along with adjacent amino acids to promote context-specific antibody generation. Post-immunization, repeated booster doses stimulate antibody diversity within the antiserum. Isolation of methylation-specific antibodies relies on affinity-based separation. Crude antiserum undergoes chromatography using columns functionalized with the immobilized methylated peptide. Non-binding antibodies are removed through rigorous washing, while retained antibodies targeting the methylated epitope are later recovered via elution buffers that weaken antigen-antibody binding. This yields a purified antibody pool with elevated specificity for the intended methylation mark.

Phage Display Strategy for Anti-Methylation Monoclonal Antibody Discovery

Phage display enables in vitro monoclonal antibody selection by linking antibody fragment genes to bacteriophage surface proteins. A diverse scFv or Fab library displayed on phages undergoes iterative screening against biotinylated methylated peptides. Target-bound phages are isolated using streptavidin-coated substrates, while non-interacting phages are discarded. Captured phages infect bacterial hosts for propagation, and subsequent selection rounds apply escalating stringency to favor high-affinity binders. Post-enrichment, monoclonal candidates are evaluated for preferential binding to methylated versus unmethylated peptides. Genetic sequences from validated clones are then reformatted into full-length antibodies or alternative therapeutic constructs.

Hybridoma Strategy for Anti-Methylation Monoclonal Antibody Discovery

Hybridoma technology combines immunized murine splenocytes with immortal myeloma cells to generate stable antibody-producing cell lines. Mice immunized with methylated antigens develop B lymphocytes expressing target-specific antibodies. Post-fusion, hybridomas are selected using media formulations that eliminate non-hybrid cells. Surviving clones undergo high-throughput screening via ELISA or analogous methods to identify those secreting methylation-specific antibodies. Positive clones undergo subcloning to ensure genetic uniformity before large-scale antibody production. This classical approach balances established reliability with the capacity for tailored monoclonal antibody development.

Types of PTM

In addition to the methylation-specific antibody, Creative Biolabs offers a comprehensive suite of custom PTM-specific antibody production services.

Service Highlights

Precise Specificity Engineering: We create antibodies that precisely differentiate methylation patterns, including mono-, di-, and tri-methyl lysine variants, along with mono-, asymmetric di-, and symmetric di-methyl arginine forms.

Advanced Technology Platforms: Our platform combines phage display with hybridoma technology to isolate high-affinity antibodies with diverse binding profiles across target epitopes.

Flexible Antibody Solutions: We develop custom monoclonal and affinity-purified polyclonal antibodies, optimized for your experimental needs in epigenetics and beyond.

Rigorous Validation and Characterization: Every antibody undergoes thorough validation for specificity and binding strength, with full performance data included to support reproducibility in epigenetic assays.

Immunogen Optimization Expertise: Our scientists design immunogens—from modified peptides to protein constructs—to maximize antibody specificity and reactivity during immunization.

Cases

Monospecific Anti-Methylation Polyclonal Antibody Production

• QC analysis certificate of the synthesized methylated peptide

• Specificity test of anti-methylated peptide polyclonal antibody by Dot blot

Q&A

Q: What factors influence the specificity of your methylation antibodies?

A: Antibody specificity derives from multiple coordinated strategies. Immunogens are precision-engineered, incorporating methylated residues flanked by sequence motifs that optimize structural context. Post-immunization, systematic screening via ELISA and dot blotting eliminates cross-reactivity with non-methylated analogs or unrelated post-translational modifications. Validation extends to independent analytical techniques, such as mass spectrometry, to verify exclusive binding to the intended methylated target.

Q: What antigen formats do you recommend for generating methylation-specific antibodies?

A: Antigen design adapts to methylation site complexity and experimental goals. Short synthetic peptides featuring the methylated residue within their native sequence are commonly conjugated to carrier proteins like KLHC or BSA. For targets requiring tertiary structure recognition, recombinant methylated protein domains may serve as immunogens. Our experts collaborate with our customers to select antigen configurations balancing immunogenicity and epitope accessibility.

Q: What are the advantages of using monoclonal and polyclonal antibodies for methylation studies?

A: Monoclonal antibodies deliver uniform epitope recognition across production batches, ideal for standardized assays requiring precise methylation-state discrimination. Polyclonal preparations, by contrast, exhibit multi-epitope avidity that may enhance detection sensitivity in certain applications. Selection hinges on experimental priorities: monoclonal antibodies suit quantitative analyses, while polyclonals may excel in target enrichment workflows. Customization options exist for both antibody classes.

Q: Can you provide scale-up production of selected antibody clones?

A: Our facilities support full-scale manufacturing of validated clones, delivering yields from milligrams to grams. Production workflows incorporate fermentation optimization and chromatography-based purification tailored to each antibody's biochemical properties. Post-production, we offer formulation adjustments—including buffer exchanges and concentration standardization—to meet application-specific stability and compatibility requirements.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.