CBLL2 Antibody

What is CBLL2 and how does it relate to other molecular targets?

CBLL2 (also known as ZNF645, HAKAIL, or CT138) is a 49kDa protein that functions as part of the Casitas B-lineage Lymphoma family of E3 ubiquitin ligases . While distinct from the well-characterized CBL (p120) protein, CBLL2 shares structural homology with other family members that regulate immune cell function . The protein is encoded by the ZNF645 gene with UniProt ID Q8N7E2 and NCBI reference sequence NP_689790 . Unlike the classical CBL protein that has been extensively studied in various cancer cell lines including MCF-7, K562, and Raji cells , CBLL2's functional characterization remains more limited in the scientific literature.

What experimental applications are validated for commercial CBLL2 antibodies?

Current commercial CBLL2 antibodies have been validated primarily for Western Blot (WB) applications, with recommended titrations between 0.2-1 μg/ml for optimal detection . While immunodetection represents the core application, researchers should note that:

• Positive controls such as MCF7 cell lysate have been validated for detection

• Cross-reactivity has been documented with canine and yeast samples

• The antibody specifically recognizes the synthetic peptide sequence: LSPQFTQTDAMDHRRWPAWKRLSPCPPTRSPPPSTLHGRSHHSHQRRHRR

Unlike antibodies for related proteins like CBL, which have been validated across multiple applications and detection methods including Simple Western™ , CBLL2 antibodies currently have more limited application validation.

What are critical storage and handling parameters for maintaining CBLL2 antibody performance?

To maintain optimal reactivity and specificity of CBLL2 antibodies, adhere to the following research-validated protocols:

Antibody performance is significantly affected by buffer composition, with current formulations utilizing 1x PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose . This buffer composition maintains stability while preventing microbial contamination during storage periods. Researchers should avoid repeated freeze-thaw cycles by preparing appropriately sized aliquots for experimental use.

How can researchers validate the specificity of CBLL2 antibodies in experimental systems?

Validating CBLL2 antibody specificity requires a multi-faceted approach beyond simple immunoblotting. Based on established protocols for similar antibodies:

• Peptide competition assays: Pre-incubate antibody with excess immunizing peptide (LSPQFTQTDAMDHRRWPAWKRLSPCPPTRSPPPSTLHGRSHHSHQRRHRR) to confirm signal reduction in positive control samples .

• Molecular weight verification: Confirm detection of the expected 49kDa band in Western blot applications .

• Knockout/knockdown validation: Generate CRISPR/Cas9 knockout or siRNA knockdown of CBLL2/ZNF645 to verify signal loss. This approach has proven valuable in validating related E3 ubiquitin ligase antibodies .

• Cross-reactivity assessment: Test against recombinant proteins from the CBL family to confirm specificity, particularly important given structural similarities within this protein family .

• Multiple detection methods: When possible, validate across orthogonal techniques beyond Western blotting to strengthen confidence in specificity.

What methodological considerations are critical when designing experiments to study CBLL2's role in cellular pathways?

When investigating CBLL2's role in cellular signaling and function, researchers should consider:

• Context-dependent expression: E3 ubiquitin ligases like CBLL2 often demonstrate context-dependent expression patterns across cell types. Preliminary experiments should establish baseline expression levels in experimental models .

• Functional redundancy: As with other CBL family members, functional redundancy may mask phenotypes in single-knockout systems. Consider combinatorial approaches targeting multiple family members simultaneously .

• Substrate identification: For studying CBLL2's enzymatic activity, utilize immunoprecipitation coupled with mass spectrometry to identify potential ubiquitination substrates, similar to approaches used for CBL .

• Activation requirements: E3 ubiquitin ligases typically require specific activation signals. When studying CBLL2 activity, consider experimental stimuli that might trigger its activation, potentially including CD28 costimulation pathways as observed with related family members .

• Temporal dynamics: Ubiquitination is a dynamic process; time-course experiments are essential for capturing transient interactions and modifications.

How does the design of anti-CBLL2 antibodies impact their performance in different experimental contexts?

Antibody design significantly impacts experimental performance and reliability. For CBLL2 antibodies:

• Epitope selection: Current CBLL2 antibodies target the middle region of the protein , which may impact accessibility in certain applications. For complex or native conformations, alternative epitopes might be required.

• Polyclonal versus monoclonal considerations: Available CBLL2 antibodies are polyclonal , which provides broader epitope recognition but potentially increased batch variability compared to monoclonal alternatives.

• Cross-species reactivity: Current antibodies show predicted reactivity with canine and yeast samples in addition to human targets . This cross-reactivity should be considered when designing experiments in different model systems.

• Conformational recognition: Recent advances in antibody development highlight the importance of conformational epitopes. As seen with SARS-CoV-2 antibodies, naive B cells often target epitopes exposed in open or altered conformations . This principle may apply to CBLL2 antibody recognition of native versus denatured protein forms.

What are common technical challenges when working with CBLL2 antibodies and how can they be addressed?

Researchers frequently encounter several technical challenges when working with CBLL2 antibodies:

• High background signal: This can be addressed by:

  • Optimizing blocking conditions (5% non-fat milk vs. BSA)

  • Increasing wash duration and stringency

  • Titrating primary antibody concentration (start with 0.2 μg/ml and adjust based on signal-to-noise ratio)

  • Using fresh transfer buffers and membranes

• Multiple bands: To resolve multiple band detection:

  • Compare results with positive control lysates (e.g., MCF7)

  • Verify with alternative antibodies targeting different epitopes

  • Conduct peptide competition assays to identify specific bands

  • Consider post-translational modifications that may alter apparent molecular weight

• Weak signal detection: Enhance signal by:

  • Increasing protein loading (optimize between 20-50μg)

  • Extending primary antibody incubation (overnight at 4°C)

  • Utilizing signal enhancement systems (e.g., biotin-streptavidin)

  • Testing alternative extraction buffers to improve target solubilization

• Inconsistent results across experiments: Improve reproducibility by:

  • Aliquoting antibody to minimize freeze-thaw cycles

  • Standardizing lysate preparation protocols

  • Maintaining consistent incubation times and temperatures

  • Documenting lot numbers and conducting lot-to-lot validation

How can computational approaches enhance CBLL2 antibody development and application?

Computational methods are increasingly valuable for antibody development and optimization, particularly for challenging targets like CBLL2:

• Epitope prediction and optimization: Computational algorithms can identify optimal epitopes based on:

  • Surface accessibility

  • Sequence conservation

  • Secondary structure prediction

  • Antigenicity scoring

• Specificity enhancement: Recent advances in biophysics-informed modeling allow for:

  • Identification of distinct binding modes associated with specific ligands

  • Prediction of cross-reactivity with related protein family members

  • Design of variants with customized specificity profiles

• Structure-guided optimization: Using homology modeling based on related CBL family structures to:

  • Identify critical binding determinants

  • Guide antibody engineering for improved performance

  • Predict potential conformational epitopes

• High-throughput sequence analysis: Next-generation sequencing data can be leveraged to:

  • Analyze antibody selection experiments

  • Identify enriched complementarity-determining regions (CDRs)

  • Design libraries with improved properties

As demonstrated in recent research, "biophysics-informed models trained on experimentally selected antibodies can associate distinct binding modes with potential ligands, enabling prediction and generation of specific variants beyond those observed in experiments" .

How might CBLL2 antibodies be leveraged in studying immune regulation and cancer biology?

Based on structural and functional homology with other CBL family proteins, CBLL2 antibodies may provide valuable insights into:

• T cell signaling regulation: Similar to Cbl-b, CBLL2 may function as a regulator of T cell activation. Antibodies can be used to:

  • Track protein expression during T cell activation

  • Identify interaction partners through co-immunoprecipitation

  • Monitor ubiquitination events following receptor stimulation

• Cancer immunotherapy applications: E3 ubiquitin ligases like Cbl-b have emerged as promising targets for cancer immunotherapy:

  • CBLL2 antibodies could help characterize expression in tumor microenvironments

  • Monitor changes following experimental treatments

  • Serve as research tools for developing targeted therapies

• Signaling pathway analysis: Using CBLL2 antibodies in combination with phospho-specific antibodies to:

  • Dissect signaling networks in normal and pathological states

  • Understand temporal dynamics of protein-protein interactions

  • Characterize post-translational modifications that regulate activity

• Therapeutic target validation: As observed with Cbl-b, which "regulates both innate and adaptive immune cells, ultimately promoting an immunosuppressive tumor microenvironment" , CBLL2 antibodies can help determine whether this protein represents a valid therapeutic target through mechanistic studies.

How are emerging antibody engineering technologies impacting research applications for targets like CBLL2?

Recent technological advances are transforming antibody development approaches for complex targets like CBLL2:

• Phage display optimization: By combining high-throughput sequencing with computational analysis, researchers can now:

  • Identify antibodies with customized specificity profiles

  • Generate variants with either high specificity for CBLL2 or controlled cross-reactivity with related family members

  • Mitigate experimental artifacts and biases in selection experiments

• Single B cell sequencing: This approach enables:

  • Isolation of naturally occurring antibodies with desired properties

  • Identification of paired heavy and light chain sequences

  • Analysis of sequence diversity and maturation pathways

• Structure-guided design: Computational approaches using:

  • Energy functions to optimize binding interfaces

  • Simultaneous optimization of multiple binding modes

  • Generation of cross-specific or highly selective antibodies

• CRISPR-based validation: Advanced validation techniques using:

  • Genetic knockout of target proteins

  • Epitope tagging of endogenous proteins

  • Domain-specific modifications to map binding regions

These technologies provide researchers with unprecedented control over antibody properties, allowing the design of CBLL2 antibodies with predefined binding profiles that can be "either cross-specific, allowing interaction with several distinct ligands, or specific, enabling interaction with a single ligand while excluding others" .

What novel experimental approaches might overcome current limitations in CBLL2 antibody applications?

To address current limitations, researchers might consider:

• Nanobody and single-domain antibody development: These smaller antibody formats offer:

  • Improved access to sterically hindered epitopes

  • Enhanced tissue penetration for in vivo applications

  • Opportunities for multispecific targeting approaches

• Proximity-dependent labeling: Using CBLL2 antibodies conjugated to enzymes like:

  • TurboID or miniTurbo for biotin labeling of proximal proteins

  • APEX2 for peroxidase-mediated labeling

  • Photo-crosslinking for capturing transient interactions

• Live-cell imaging applications: Developing:

  • Intrabodies for tracking CBLL2 dynamics in living cells

  • Split fluorescent protein complementation to visualize interactions

  • FRET-based sensors to monitor conformational changes

• Combinatorial targeting strategies: Creating:

  • Bispecific antibodies targeting CBLL2 and interacting proteins

  • Antibody-drug conjugates for targeted protein degradation

  • Immunoprecipitation-optimized variants for complex purification

These approaches could transform CBLL2 antibodies from simple detection tools into sophisticated research instruments for elucidating protein function and regulation in complex biological systems.