CBL4 Antibody

What is CBLN4 and what cellular functions does it perform?

CBLN4 (Cerebellin-4) is a 26-35 kDa secreted glycoprotein member of the C1q/TNF Superfamily. Mature human CBLN4 consists of 174 amino acids (aa 28-201) and contains two N-terminal cysteines that facilitate homohexamer formation, along with a C-terminal C1q domain (aa 66-201) that promotes homotrimer formation. In testicular tissue, CBLN4 likely promotes cell differentiation, while in neural tissues, it may participate in synapse formation through interactions with neurexin. CBLN4 can also potentially multimerize with other cerebellin-related molecules (CBLN1-3). The protein shows remarkable conservation across species, with mature human CBLN4 sharing 99% amino acid identity with mouse CBLN4 .

In which tissues and cell types is CBLN4 primarily expressed?

CBLN4 expression is primarily observed in developing Sertoli cells in testicular tissue and in select neurons within specific brain regions, including the dorsal raphe, entorhinal cortex, and arcuate nucleus. Immunohistochemistry studies have detected CBLN4 in human hypothalamus, where specific staining was localized to neuronal cell bodies and processes. This expression pattern suggests CBLN4 plays specialized roles in both reproductive and neurological systems .

What applications are CBLN4 antibodies validated for?

Based on the provided search results, CBLN4 antibodies have been validated for several experimental applications:

• Western Blot (WB): For detecting CBLN4 protein in cell and tissue lysates

• Immunohistochemistry (IHC): For visualizing CBLN4 in tissue sections, as demonstrated in human hypothalamus samples

• Immunocytochemistry (ICC): For cellular localization studies

Specific antibodies like the Sheep Anti-Human Cerebellin-4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6740) have been validated for IHC on paraffin-embedded sections using appropriate antigen retrieval methods .

What are the optimal conditions for CBLN4 antibody storage and handling?

For optimal storage and handling of CBLN4 antibodies, researchers should follow these guidelines:

• Storage Temperature:

  • Long-term storage (up to 12 months): -20°C to -70°C as supplied

  • Short-term storage (up to 1 month): 2-8°C under sterile conditions after reconstitution

  • Medium-term storage (up to 6 months): -20°C to -70°C under sterile conditions after reconstitution

• Handling Precautions:

  • Use a manual defrost freezer to avoid temperature fluctuations

  • Avoid repeated freeze-thaw cycles which can compromise antibody functionality

  • Maintain sterile conditions during reconstitution and subsequent handling

  • Allow antibodies to equilibrate to room temperature before opening vials

How should I determine the optimal dilution of CBLN4 antibody for my specific application?

Determining the optimal dilution for CBLN4 antibodies requires systematic testing:

• For Immunohistochemistry:

  • Start with manufacturer-recommended concentrations (e.g., 10 μg/mL for AF6740)

  • Perform a dilution series (e.g., 5, 10, 15, 20 μg/mL) to identify the concentration that maximizes specific signal while minimizing background

  • Include appropriate negative controls (isotype control or no primary antibody)

• For Western Blot:

  • Begin with a 1:500 to 1:1000 dilution range

  • Test multiple dilutions in a pilot experiment

  • Select the dilution that provides clear band visualization at the expected molecular weight (26-35 kDa) with minimal non-specific binding

Remember that optimal dilutions should be determined by each laboratory for each application, as noted in the product documentation .

What gel percentage should I use for optimal resolution of CBLN4 in Western blot experiments?

For optimal resolution of CBLN4 (26-35 kDa) in Western blot experiments, select the gel percentage based on the protein's molecular weight:

Since CBLN4 falls within the 26-35 kDa range, a 10-12% Tris-Glycine gel would provide optimal resolution. Alternatively, for experiments involving multiple proteins of varying sizes, a 4-20% Tris-Glycine gradient gel can provide good resolution across a broader molecular weight range .

What controls should be included when using CBLN4 antibodies for detecting post-translational modifications?

When investigating post-translational modifications (PTMs) of CBLN4, include these essential controls:

• Positive Controls:

  • Lysates from cell lines with known CBLN4 expression (based on literature or manufacturer data)

  • Recombinant CBLN4 protein with documented PTMs

  • Samples from tissues with high CBLN4 expression (e.g., hypothalamus)

• Negative Controls:

  • CBLN4 knockout/knockdown cell lines or tissues

  • Samples treated with phosphatases or deglycosylation enzymes (for phosphorylation or glycosylation studies)

  • Isotype control antibodies

• Treatment Controls:

  • Samples subjected to conditions known to modify CBLN4 (if documented)

  • Time-course experiments to capture dynamic changes in modification

For specific PTM studies, consult resources like PhosphoSitePlus® for information on CBLN4 modification residues and their functional significance. This approach ensures confident identification of genuine PTM signals versus potential artifacts .

How can I optimize CBLN4 detection in hypothalamus tissue sections?

For optimal detection of CBLN4 in hypothalamus tissue sections, implement this specialized protocol:

• Tissue Preparation:

  • Use properly fixed (immersion-fixed) paraffin-embedded sections

  • Ensure sections are cut at appropriate thickness (4-6 μm)

• Antigen Retrieval:

  • Perform heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic

  • Maintain precise temperature control during retrieval

• Antibody Application:

  • Use validated antibodies (e.g., Sheep Anti-Human Cerebellin-4)

  • Apply at 10 μg/mL concentration

  • Incubate overnight at 4°C for optimal binding

• Detection System:

  • Employ a sensitive detection system like HRP-DAB

  • Counterstain with hematoxylin to visualize cellular context

  • Evaluate specific staining in neuronal cell bodies and processes

• Controls:

  • Include sections without primary antibody

  • Use isotype control antibodies

  • Compare with sections from other brain regions as reference

What strategies can be employed to study CBLN4 interactions with neurexin and other binding partners?

To investigate CBLN4 interactions with neurexin and other potential binding partners, consider these advanced approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-CBLN4 antibodies to pull down protein complexes

  • Analyze precipitated proteins by Western blot or mass spectrometry

  • Validate with reciprocal Co-IP using antibodies against suspected binding partners

• Proximity Ligation Assay (PLA):

  • Apply paired antibodies against CBLN4 and potential binding partners

  • Visualize protein-protein interactions in situ with subcellular resolution

  • Quantify interaction signals across different experimental conditions

• Bioluminescence Resonance Energy Transfer (BRET):

  • Generate fusion constructs of CBLN4 and binding candidates with appropriate tags

  • Measure energy transfer as indication of protein proximity

  • Establish binding kinetics through concentration-dependent experiments

• Surface Plasmon Resonance (SPR):

  • Immobilize purified CBLN4 or binding partners on sensor chips

  • Measure real-time binding kinetics and affinity constants

  • Evaluate effects of mutations on interaction strength

These techniques provide complementary data on CBLN4 interactions, enabling comprehensive characterization of its binding network and functional significance .

How can I address non-specific binding issues when using CBLN4 antibodies?

To minimize non-specific binding when using CBLN4 antibodies:

• Antibody Selection:

  • Use antibodies validated specifically for your application

  • Consider affinity-purified antibodies (e.g., Affinity-purified Polyclonal Antibody)

  • Check cross-reactivity data in manufacturer documentation

• Blocking Optimization:

  • Test different blocking agents (BSA, casein, normal serum)

  • Increase blocking time or concentration

  • Use blocking serum from the same species as the secondary antibody

• Buffer Adjustments:

  • Add 0.1-0.5% Triton X-100 or Tween-20 to reduce hydrophobic interactions

  • Optimize salt concentration in wash buffers (150-500 mM NaCl)

  • Consider adding 5-10% normal serum to antibody dilution buffer

• Antibody Dilution:

  • Further dilute primary antibody if background is high

  • Perform a systematic dilution series to find optimal concentration

  • Reduce secondary antibody concentration independently

• Incubation Conditions:

  • Extend washing steps (number and duration)

  • Perform antibody incubation at 4°C for longer periods

  • Filter antibody solutions before use

What factors should I consider when comparing results from different CBLN4 antibody clones?

When comparing results obtained with different CBLN4 antibody clones, consider these critical factors:

• Epitope Recognition:

  • Different clones may target distinct epitopes on CBLN4

  • Epitope accessibility can vary across experimental conditions

  • Structural modifications or protein interactions may mask specific epitopes

• Antibody Format and Species:

  • Polyclonal vs. monoclonal antibodies provide different coverage

  • Host species affects background in certain applications

  • Different isotypes may have varying tissue penetration properties

• Validation Parameters:

  • Check if antibodies are validated for your specific application

  • Review published literature using each antibody

  • Consider performing side-by-side comparison in your system

• Sample Preparation Compatibility:

  • Some antibodies work better with certain fixation methods

  • Antigen retrieval requirements may differ between clones

  • Denaturing vs. native conditions may affect epitope recognition

• Analysis Metrics:

  • Document observed molecular weights for each antibody

  • Compare signal-to-noise ratios across antibodies

  • Note differences in subcellular localization patterns

Creating a systematic comparison table with these parameters helps reconcile disparate results and select the most appropriate antibody for specific research questions .

How can I validate the specificity of a CBLN4 antibody in my experimental system?

To comprehensively validate CBLN4 antibody specificity:

• Molecular Approaches:

  • CBLN4 knockdown/knockout: Confirm signal reduction/elimination

  • Overexpression studies: Demonstrate increased signal intensity

  • Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Technical Controls:

  • Multiple antibodies targeting different CBLN4 epitopes

  • Secondary-only controls to assess non-specific binding

  • Isotype controls to evaluate background

• Cross-Reactivity Assessment:

  • Test on tissues/cells known to be negative for CBLN4

  • Analyze species cross-reactivity if working with non-human models

  • Check for signal in off-target locations inconsistent with known biology

• Method Triangulation:

  • Confirm findings using orthogonal techniques (Western blot, IHC, IF)

  • Compare protein detection with mRNA expression (qPCR, in situ hybridization)

  • Use mass spectrometry to confirm identity of detected proteins

• Expected Characteristics:

  • Verify molecular weight (26-35 kDa)

  • Confirm expression pattern matches literature (hypothalamus, testis)

  • Validate subcellular localization aligns with known biology

How can CBLN4 antibodies be used to investigate its role in synapse formation and neurological disorders?

CBLN4 antibodies can be strategically employed to explore its role in synapse formation and neurological disorders through these approaches:

• High-Resolution Localization Studies:

  • Use immunofluorescence with super-resolution microscopy to map CBLN4 distribution at synapses

  • Perform co-localization studies with pre/post-synaptic markers to determine precise positioning

  • Analyze temporal expression during synaptogenesis in development or following injury

• Functional Activity Correlation:

  • Combine CBLN4 immunostaining with activity-dependent markers

  • Analyze CBLN4 levels in response to neuronal stimulation or silencing

  • Correlate CBLN4 expression with electrophysiological recordings

• Disease State Analysis:

  • Compare CBLN4 expression patterns in normal vs. pathological human brain samples

  • Evaluate CBLN4 levels in animal models of neurological disorders

  • Assess post-translational modifications of CBLN4 in disease conditions

• Therapeutic Intervention Studies:

  • Monitor CBLN4 changes following pharmacological treatments

  • Use neutralizing antibodies to block CBLN4 function in experimental models

  • Track CBLN4 dynamics during recovery phases

This research can provide insights into CBLN4's potential as a biomarker or therapeutic target for synaptopathies and neurodevelopmental disorders .

What approaches can be used to study the multimerization of CBLN4 with other cerebellin family members?

To investigate CBLN4 multimerization with other cerebellin family members (CBLN1-3), implement these specialized techniques:

• Biochemical Characterization:

  • Size exclusion chromatography to separate different multimeric forms

  • Blue native PAGE to preserve native protein complexes

  • Chemical crosslinking followed by immunoprecipitation with CBLN4-specific antibodies

• Structural Studies:

  • Use multiple antibodies targeting different domains to map interaction regions

  • Employ Förster resonance energy transfer (FRET) to measure proximity between labeled cerebellin proteins

  • Apply hydrogen-deuterium exchange mass spectrometry to identify interaction interfaces

• Cellular Imaging:

  • Multi-color immunofluorescence to visualize co-localization of CBLN proteins

  • Split-GFP complementation assays to detect direct interactions

  • Live-cell imaging to track dynamics of complex formation

• Functional Analysis:

  • Compare signaling outcomes of homomeric vs. heteromeric complexes

  • Assess binding preferences to neurexins or other partners

  • Evaluate developmental regulation of complex composition

These approaches will help elucidate how CBLN4's ability to multimerize with other family members contributes to its functional diversity in different cellular contexts .

How can CBLN4 antibodies be used to investigate its role in Sertoli cell development and testicular function?

To investigate CBLN4's role in Sertoli cell development and testicular function using antibodies:

• Developmental Profiling:

  • Perform immunohistochemistry across different developmental stages

  • Quantify CBLN4 expression changes during critical windows of Sertoli cell maturation

  • Compare with known Sertoli cell differentiation markers

• Functional Manipulation Studies:

  • Use neutralizing antibodies in ex vivo testicular cultures

  • Assess effects on Sertoli cell proliferation, differentiation, and function

  • Analyze consequent changes in spermatogenesis

• Signaling Pathway Analysis:

  • Combine CBLN4 immunostaining with phospho-specific antibodies for potential downstream effectors

  • Perform co-immunoprecipitation to identify binding partners in testicular tissue

  • Create signaling networks based on protein interaction data

• Pathological Investigations:

  • Evaluate CBLN4 expression in testicular biopsies from infertility patients

  • Compare CBLN4 levels across different testicular pathologies

  • Correlate CBLN4 patterns with specific functional deficits

• Mechanistic Studies:

  • Analyze CBLN4 secretion patterns from Sertoli cells

  • Investigate paracrine effects on adjacent cell types

  • Identify receptors mediating CBLN4 actions in testicular tissue

This systematic approach will provide insights into CBLN4's function in reproductive biology and potential implications for fertility research .