Phosphorylation detection commonly employs antibodies engineered for phosphoepitope recognition. Building upon decades of investigation into phospho-recognition motifs combined with High-Affi™ platform implementation, Creative Biolabs offers high-affinity antibodies targeting this extensively characterized post-translational modification (PTM), phosphorylation.

Background

Phosphorylation involves covalent PO₄³⁻ group incorporation into proteins or other biomolecules, representing a biologically indispensable PTM occurring universally. Cellular phosphorylation dynamics emerge through regulated kinase-phosphatase equilibrium, with such reversible modifications impacting approximately 30% of proteomic constituents. These dynamic alterations exert regulatory control over multifaceted biological phenomena spanning subcellular localization, structural remodeling events, metabolic coordination, catalytic function modulation, and protein-protein interactions. Pathological phosphorylation irregularities demonstrate strong associations with neurodegeneration, oncogenic progression, metabolic dysregulation, cardiovascular pathologies, and immune system malfunctions.

Fig.1 The phosphorylation process and the potential functions of phosphorylation.¹

Phospho-sites typically manifest as multiple discrete residues within individual proteins, with kinase specificity dictating modification patterns. Eukaryotic systems principally exhibit phosphorylation on hydroxyl-containing residues, like serine, threonine, and tyrosine, though histidine modification occasionally occurs. Prokaryotic phosphorylation demonstrates broader residue selectivity, including basic amino acids. Serine represents the most frequent phosphorylation site, followed by threonine. Despite constituting <1% of total modifications, tyrosine phosphorylation holds particular biological significance as an initiator of signaling cascades in higher organisms.

Antibody Types

Our services encompass the production of two primary types of phosphorylation-specific antibodies, each leveraging the specificity of antibodies to target phosphorylated proteins:

Phosphorylation Polyclonal Antibodies: Phosphorylation polyclonal antibodies are generated from multiple B-cell clones and recognize various epitopes on the target protein. This polyclonal nature often provides a stronger signal due to their ability to bind to multiple sites on the phosphorylated protein, which can be advantageous in assays where sensitivity is paramount for detecting phosphorylation events.

Phosphorylation Monoclonal Antibodies: Phosphorylation monoclonal antibodies, derived from a single B-cell clone, offer exceptional specificity, recognizing only a single, defined epitope on the phosphorylated target protein. This high degree of specificity makes them invaluable for applications requiring precise identification and quantification of specific phosphorylation events.

Discovery Strategy

Monospecific Anti-Phosphorylation Polyclonal Antibody Production

This approach focuses on creating phosphosite-specific polyclonal antibodies through systematic antigen engineering. The workflow initiates with computationally-guided peptide design, prioritizing sequences containing target phosphorylation motifs flanked by residues essential for antibody selectivity. Synthetic phosphopeptides undergo stabilization treatments before conjugation to immunogenic carriers such as KLH or BSA. Host immunization protocols employ iterative serum monitoring via comparative ELISA against phospho- and non-phospho-peptides. Antibody purification implements a multi-step affinity chromatography methodology: initial phosphopeptide matrix isolation precedes subtractive chromatography using non-phosphorylated analogues. This bidirectional purification cascade produces antibody pools demonstrating superior target discrimination capacity.

Phage Display Strategy for anti-Phosphorylation Monoclonal Antibody Discovery

Phage display enables the in vitro selection of high-affinity monoclonal antibodies for phosphopeptides. A library of antibody fragments (e.g., scFv, Fab) is displayed on bacteriophages, which are incubated with a biotinylated target phosphopeptide. Phages with binding antibodies are retained, while non-binding ones are washed away. After several rounds of selection, high-affinity clones are isolated and their specificity is tested against both phosphorylated and unphosphorylated peptides. This method offers advantages such as generating antibodies for non-immunogenic targets, high-throughput screening, and direct isolation of recombinant antibody genes for further production.

Hybridoma Strategy for Anti-Phosphorylation Monoclonal Antibody Discovery

The hybridoma platform enables production of clonal phosphoepitope-recognizing antibodies through cellular fusion techniques. Murine immunization regimens utilize optimized phosphopeptide-carrier conjugates to stimulate epitope-focused B-cell responses. Post-immunization, splenocyte-myeloma cell fusion generates immortalized antibody-producing hybrids. Clonal expansion precedes multi-tiered screening processes combining phosphopeptide-specific ELISA with cross-reactivity assessments against non-phosphorylated protein. Secondary validation incorporates immunoblotting and tissue staining to exclude non-specific clones. This iterative selection protocol identifies hybridomas secreting antibodies with stringent phosphorylation dependency, ensuring batch-to-batch consistency for diagnostic and research applications.

Types of PTM

We provide phospho-pan/phospho-site specific antibodies and antibodies targeting a variety of PTMs, as shown in the table below.

Service Highlights

Reliable Antibody Generation: Our demonstrated success metrics in antibody generation derive from protocol optimization and extensive expertise across our protein engineering specialists. These established methodologies enable reliable production of functionally validated antibodies.

Tailored Antigen Design: The service features adaptable antigen design incorporating sequence optimization protocols that mirror phosphorylation site architecture. Peptide synthesis integrates structural modifications enhancing antigenic verisimilitude, ensuring precise antibody-epitope compatibility for target-specific recognition.

Comprehensive Quality Assurance: Quality control systems implement continuous monitoring during antibody development phases. Final validation incorporates parallel testing matrices using epitope-specific ELISA quantification, immunoblot cross-reactivity profiling, to confirm application-ready performance specifications.

Dedicated Expert Support: Our scientific team delivers continuous consultation encompassing experimental design optimization through data interpretation phases. Specialists provide actionable insights regarding host species compatibility, immunization regimen calibration, and validation parameter establishment for complete project lifecycle management.

Efficient and Cost-Effective Solutions: Cost-competitive service leverages optimized production pipelines and bulk reagent procurement strategies. Accelerated timelines employ parallel processing protocols and just-in-time manufacturing principles, delivering batch-to-batch consistent antibodies within predefined budgetary parameters.

Cases

We have developed a variety of phosphorylation antibody products for customers, and the case and the high-specificity results of the obtained antibodies are shown in the figure below.

Case 1: Monospecific Anti-Phosphorylation Polyclonal Antibody Production

• QC analysis certificate of the synthesized phosphorylated peptide

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

Q&A

Q: What is the typical timeline for completing a phosphorylation-specific antibody project?

A: Project duration fluctuates based on variables such as antibody format (polyclonal versus monoclonal) and target complexity. Our team collaborates with clients to define feasible schedules, maintaining ongoing communication regarding progress at each developmental phase.

Q: What validation methods confirm antibody specificity for phosphorylated targets?

A: Specificity assurance integrates multi-layered quality control: antigen sequences are optimized to minimize cross-reactivity, purification incorporates negative selection against non-phosphorylated analogs, and post-production validation combines ELISA, WB, and competitive binding assays with phosphorylated/non-phosphorylated peptide pairs.

Q: Is expert guidance available for developing effective antigen designs?

A: Our specialists support antigen optimization by analyzing peptide length, phosphorylation site accessibility, and carrier protein compatibility. Recommendations address structural modifications to enhance immunogenicity while minimizing off-target epitope recognition, informed by decades of empirical success in antibody generation.

Q: What sample types are suitable for testing with your phospho-specific antibodies?

A: Validated applications include cell lysates, tissue homogenates, and recombinant protein preparations. Antibody suitability for specific sample matrices is empirically confirmed during validation, with protocols available for optimizing buffer conditions, fixation methods, and antigen retrieval to address challenging sample types.

Q: Are performance assurances provided for purchased antibodies?

A: All products undergo application-specific validation, with guarantees covering epitope specificity and functionality in agreed-upon assays. Customized commitments are negotiable based on project requirements, supported by technical documentation detailing validation parameters. Consult our team for specific guarantee policies with experimental objectives.

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