Post-translational modifications (PTMs) are critical regulators governing multifaceted biological processes. Research progress in this domain frequently encounters limitations stemming from inadequate detection tools, particularly due to the low antigenic potential and ubiquitous nature of most PTM epitopes. In response to these methodological constraints, Creative Biolabs has developed the High-Affi™ technology, a service platform for generating antibodies with superior target discrimination against protein PTMs.

PTM Background

PTMs constitute enzymatically mediated structural changes implemented after polypeptide chain assembly. These biochemical transformations primarily involve covalent addition of molecular moieties, exemplified by phosphate group attachment and carbohydrate conjugation. Such modifications fundamentally participate in cellular physiology through roles in differentiation mechanisms, proteostasis regulation, intracellular communication networks, transcriptional modulation, and intermolecular recognition events. Considering their pervasive involvement in both homeostatic cellular functions and pathological states, precise characterization of PTM patterns holds critical significance for advancing core biological knowledge while informing therapeutic intervention approaches across multiple disease contexts.

Fig.1 Overview of the most common PTMs.¹

Types of PTM

Creative Biolabs provides specific antibody discovery services on a variety of post-translational modifications:

Antibody Types

Creative Biolabs offers two main antibody formats for PTM-specific antibody discovery:

• Polyclonal antibodies

These are a mixture of antibodies produced by different immune cells in an animal in response to a specific antigen. They recognize various epitopes on the antigen and can be useful for detecting a wide range of PTMs. This can lead to a stronger overall signal.

• Monoclonal antibodies

These are produced from a single clone of immune cells and have a high degree of specificity for a particular epitope on the antigen. This high specificity can be advantageous for studying specific PTMs. They offer high specificity and batch-to-batch consistency.

Besides, we offer the two main categories of antibodies based on PTM specificity:

• Site-specific anti-PTM antibodies

These are highly specific antibodies that recognize a particular PTM at a specific amino acid residue within a protein. For example, an antibody that specifically binds to a protein only when Lysine 42 is acetylated.

• Pan anti-PTM antibodies

These antibodies recognize a specific type of PTM regardless of the protein or the specific amino acid residue on which it occurs. For example, an anti-phosphorylation antibody that binds to any phosphorylated serine, threonine, or tyrosine residue.

Common animal sources

Rabbits: Rabbits are a common source for polyclonal antibodies.

Mouse: Mouse is commonly used for generating monoclonal antibodies.

Sheep: Sheep can also be used to produce antibodies, particularly for large-scale applications.

Discovery Strategy

Our researchers have extensive experience in PTM-specific antibody discovery. We offer three different discovery strategies that we can customize based on your modification type to meet the needs of different customers.

Monospecific Anti-PTM Polyclonal Antibody Production

This strategy focuses on generating a highly specific polyclonal antibody pool by immunizing animals with a precisely designed antigen carrying the post-translational modification of interest. The resulting serum undergoes a rigorous two-step purification process involving positive selection on the modified antigen and negative selection against the unmodified counterpart. This ensures that the enriched polyclonal antibodies exhibit high sensitivity and specificity for the PTM, recognizing it within its local peptide context and potentially offering robust detection capabilities for various applications, often proving to be a cost-effective and rapid approach for initial antibody generation.

Fig.2 Workflow of monospecific anti-PTM polyclonal antibody production.

Phage Display Strategy for Anti-PTM Monoclonal Antibody Discovery

Phage display is an in vitro screening method that uses a large library of antibody fragments displayed on the surface of phages. Phage display antibody libraries can be constructed by immunizing various animals, or by directly collecting animal or human immune cells bypassing the animal immunization process. These libraries are screened against the PTM-containing antigen, allowing for the selection of high-affinity and specific binders through iterative rounds of binding, washing, and amplification. This approach offers the advantage of screening immense antibody diversity, enabling the identification of unique monoclonal antibodies against challenging targets or modifications, and allows for precise control over selection conditions, ultimately leading to fully human or humanized monoclonal antibodies suitable for a range of applications.

Fig.3 Workflow of phage display for anti-PTM monoclonal antibody discovery.

Hybridoma Strategy for Anti-PTM Monoclonal Antibody Discovery

The hybridoma technology involves immunizing animals with the PTM-specific antigen, followed by the fusion of isolated B lymphocytes with immortal myeloma cells to create hybridoma cell lines. These hybridomas are then screened to identify clones that produce monoclonal antibodies specifically recognizing the post-translational modification. This well-established method yields stable, high-quality monoclonal antibodies with consistent production and well-defined specificity, often providing robust performance in various immunological assays due to the generation of full-length immunoglobulin molecules through a natural in vivo immune response and subsequent in vitro cloning.

Fig.4 Workflow of hybridoma for anti-PTM monoclonal antibody discovery.

Cases

The following is a satisfactory experience of our customers using Creative Biolabs' PTM-specific antibody discovery service, with successful project results clearly illustrated in the following pictures.

Case 1: Monospecific Anti-Acetylation Polyclonal Antibody Production

• QC analysis certificate of the synthesized acetylated peptide

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

Case 2: 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

Case 3: Hybridoma Anti-Beta-Hydroxybutyrylated Monoclonal Antibody Production

• QC analysis certificate of the synthesized beta-hydroxybutyrylated peptide

• Specificity test of anti-beta-hydroxybutyrylated peptide monoclonal antibody by WB analysis

Q&A

Q: What varieties of PTM-targeting antibodies do you offer?

A: Our displayed services feature over 30 characterized modifications spanning phosphorylation, acetylation, methylation, and others. Clients may also receive customized solutions for rare or novel modification types. We actively pursue unconventional targets to advance PTM antibody innovation—please consult our team regarding your specialized requirements. Technical challenges in this field motivate our continuous methodological refinement.

Q: How do you ensure the specificity of the antibody for the modified site?

A: Achieving specificity constitutes our foundational priority. Antigen design incorporates precisely modified peptides paired with exhaustive counter-screening against non-modified and structurally similar epitopes. Validation employs multiple orthogonal methods, like ELISA quantification, Western blot comparisons, and IHC profiling using modified/unmodified peptides, to empirically confirm minimal off-target interactions. All specificity data undergo rigorous documentation for client review.

Q: Why select custom antibody services over off-the-shelf antibodies?

A: Though stock antibodies provide immediate accessibility, tailored development frequently yields superior experimental outcomes. We can tailor the antibody to your exact needs, including specific isoforms, species reactivity, and desired epitope. Customization extends to application-specific validation protocols that surpass generic commercial standards. This approach mitigates reproducibility concerns while aligning antibody performance with unique experimental conditions.

Q: What project information must clients provide for custom development?

A: Initiation requires comprehensive target documentation: target protein, exact PTM residue localization, adjacent sequence context (when available), intended experimental applications, and species reactivity or antibody format requirements. Detailed preliminary information enables optimized antigen design and immunization strategies. Transparent communication during this phase directly correlates with project success rates.

Q: How is quality assurance maintained throughout production?

A: Multilayer quality controls govern all workflows—from antigen purity verification (>95% HPLC certification) to affinity chromatography standardization. Batch-specific metrics include endotoxin level monitoring (<0.1 EU/mg), concentration normalization, and stability testing under multiple storage conditions. All antibodies ship with certificates detailing purity grades, specificity validation methods, and recommended usage protocols.

Q: What post-production technical assistance accompanies antibody delivery?

A: We maintain ongoing client support encompassing application troubleshooting, storage condition optimization, and experimental design consultation. Our team provides answers and guidance across experimental platforms. This collaborative relationship ensures the maximal utility of developed reagents for striving to build long-term partnerships with our clients.

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