Creative Biolabs' High-Affi™ platform enables accelerated development of antibodies targeting ubiquitination events. Our antibodies can reliably identify monoubiquitin formations, clustered multi-monoubiquitin patterns, homotypic polyubiquitin (K6, K11, K27, K29, K33, K48, K63, and linear-linked), and heterotypic polyubiquitin (mixed or branching links).

Background

Ubiquitination is a critical post-translational modification (PTM) involving covalent attachment of ubiquitin molecules (8.5 kDa regulatory proteins) to substrate proteins through isopeptide bonds. This biochemical cascade progresses through three enzymatic phases: activation by E1 enzymes, conjugation via E2 enzymes, and substrate-specific ligation mediated by E3 enzymes. Structural variations manifest as single (monoubiquitination), multiple single (multi-monoubiquitination), or chain-forming (polyubiquitination) configurations, with chains displaying either uniform linkage patterns or mixed branching. While lysine residues primarily serve as attachment sites, atypical modifications occasionally target cysteine, threonine, or serine residues.

Fig.1 Various types of ubiquitin linkages.^1,3

The dynamic equilibrium of ubiquitination arises from counteracting deubiquitination enzymes (DUBs/USPs) that enzymatically reverse these modifications. Functional consequences encompass proteasomal targeting, intracellular trafficking modulation, enzymatic activation/inhibition, and protein interaction regulation. Ubiquitination networks frequently intersect with other PTM systems. Clinical correlations link dysregulated ubiquitination pathways to neurodegenerative conditions and systemic pathologies, including malignancies, cardiovascular disorders, and metabolic diseases.

Fig.2 The ubiquitination and deubiquitination process.^1,3

Ubiquitination-specific antibodies facilitate the identification of pathological aggregates like Alzheimer-associated tau tangles. Our development pipelines combine phage display screening, hybridoma generation, and epitope-focused immunization. Available formats include scFv, Fab, and IgG antibodies raised against synthetic ubiquitin peptides, customizable by species reactivity, immunoglobulin class, and application requirements.

Fig.3 Outline of site-specific ubiquitination antibody development.^2,3

Antibody Types

We provide multiple ubiquitination-specific antibody formats, customizable by species reactivity, antibody class, and application requirements.

Monoclonal Antibodies: Characterized by singular epitope recognition, these antibodies originate from single immune cell clones, allowing for specific targeting of ubiquitin protein or conjugate complexes.

Polyclonal Antibodies: Derived from different immune cell populations, these reagents detect multiple epitopes, often enhancing signal intensity while potentially compromising specificity compared to monoclonal counterparts.

Recombinant Antibodies: Engineered through DNA recombination techniques, these antibodies ensure batch consistency and permit structural customization for specialized applications.

Antibody Fragments: Compact formats facilitating enhanced tissue penetration and adaptability for intracellular delivery or targeted therapeutic applications.

Conjugated Antibodies: Chemically coupled with detection labels (HRP, biotin) for immediate use in assays including WB, immunofluorescence, flow cytometry, and ELISA.

Linkage-Specific Antibodies: Designed to distinguish polyubiquitin chains with particular lysine linkages (e.g., K48, K63), enabling precise analysis of ubiquitination signaling pathways.

Pan-Ubiquitin Antibodies: Broad-spectrum reagents detecting ubiquitin in monomeric, polymeric, or substrate-conjugated states.

Architecture-Specific Antibodies: Specialized tools for differentiating linear versus branched ubiquitin chain configurations in functional studies.

Discovery Strategy

Monospecific Anti-Ubiquitination Polyclonal Antibody Production

We initiate monospecific polyclonal antibody development by immunizing host animals (rabbits, goats) with synthetic peptide-carrier protein conjugates mimicking ubiquitination sites. Post-immunization antisera undergoes affinity purification using immunogen-coupled matrices to isolate target-specific antibodies, yielding reagents optimized for high-sensitivity applications requiring broad epitope coverage.

Phage Display Strategy for Anti-Ubiquitination Monoclonal Antibody Discovery

Our phage display platform screens large antibody fragment libraries (scFv, Fab) against immobilized ubiquitinated antigens. Iterative binding cycles under escalating stringency conditions enrich high-affinity clones, which undergo sequencing and recombinant conversion into full-length antibodies for functional validation.

Hybridoma Strategy for Anti-Ubiquitination Monoclonal Antibody Discovery

Immunization of mice with ubiquitinated antigens precedes fusion of spleen-derived B cells with myeloma cells, creating immortalized hybridomas. Subsequent clonal screening isolates cell lines producing target-specific monoclonal antibodies, which undergo expansion for large-scale production.

Service Highlights

High-Affi™ Precision Engineering: Proprietary technology engineered to produce antibodies with exceptional selectivity for diverse ubiquitination states, including atypical and linkage-specific variants.

Custom Antigen Design: Expertise in synthesizing ubiquitinated peptides, recombinant variants, and modified conjugates to elicit targeted immune responses.

Multiplexed Discovery Platforms: Integrated access to phage display, hybridoma technology, and B cell sorting for optimal antibody generation against challenging epitopes.

Comprehensive Validation: In-house characterization covering specificity profiling, affinity measurement (KD), and application-specific testing (Western blot, IHC, IP).

Format Customization: Flexible antibody configurations (monoclonal, polyclonal, fragments) and conjugation services (fluorophores, enzymes) tailored to experimental workflows.

Types of PTM

Creative Biolabs specializes in high-specificity antibody development for multiple PTMs, including phosphorylation, glycosylation, methylation, acetylation, and ubiquitination. Our services support research, diagnostic, and industrial applications through customized PTM-specific antibody production. Beyond the ubiquitination-specific antibody, we also provide a comprehensive list of PTM-specific antibody production services of your choice.

Cases

Monospecific Anti-ubiquitination Polyclonal Antibody Production

• QC analysis certificate of the synthesized ubiquitinated peptide

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

Q&A

Q: We are interested in an antibody that specifically recognizes K48-linked polyubiquitin chains. Can you guarantee this specificity, and how do you validate it?

A: Delivering precise specificity for K48-linked chains is central to our High-Affi™ platform. We use carefully designed antigens, such as synthetic di-ubiquitin or polyubiquitin chains with exact linkage configurations. To confirm specificity, we test each antibody against competing linkages (like K63 or linear chains) and native ubiquitin via ELISA, Western blotting, and dot blot assays. Cellular lysate screening further ensures minimal off-target activity.

Q: Our research focuses on a protein ubiquitinated at multiple sites. Can you create an antibody that detects only the ubiquitinated form, regardless of the site?

A: Creating an antibody that detects only the ubiquitinated form of a protein, independent of modification sites, is technically demanding. Ubiquitination-induced structural shifts can be too subtle to expose consistent epitopes. Our approach focuses on antigens that replicate ubiquitination's broader structural effects or target conserved ubiquitin regions when bound to your protein. Feasibility depends on your target's structural data, which we'd analyze case by case.

Q: Do you have experience generating antibodies for detecting ubiquitinated proteins in FFPE tissues via IHC?

A: Yes. We've developed antibodies optimized for FFPE tissues in IHC by refining purification methods and formulations to enhance tissue penetration and epitope access post-fixation. Our workflow includes testing candidates on FFPE samples, adjusting antigen retrieval protocols, and fine-tuning staining conditions.

Q: What's the typical timeline and cost for a custom monoclonal antibody targeting a ubiquitinated epitope?

A: These projects generally run 3-6 months, though timelines shift based on target complexity, antigen immunogenicity, and screening strategy. Pricing scales with factors like required antigen quantities, the complexity of testing workflows, and final production volumes. Once we've discussed your specific needs, you'll receive a customized plan outlining phases and costs. While we aim for efficiency, some projects might need extra time, particularly if antigens underperform immunogenicity or clone selection proves trickier than anticipated.

Q: We require a large-scale, stable supply of a specific anti-ubiquitin antibody for our diagnostic kit. Can you guarantee long-term production and quality control?

A: Absolutely. Creative Biolabs has built reliable production and QC systems to ensure consistent antibody supplies for diagnostic applications. We enforce strict quality checks at every stage, from cell line upkeep to final formulation, and offer long-term agreements with predefined batch consistency standards. Sustaining cell line productivity over years requires meticulous protocol adherence, which we manage through routine monitoring.

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