CTTNBP2 Antibody

What is CTTNBP2 and why is it important in neuroscience research?

CTTNBP2 is a neuron-predominant cortactin-binding protein that is highly concentrated at dendritic spines in both cultured rat hippocampal neurons and in the mouse brain. It plays a critical role in dendritic spine formation, maintenance, and morphology. CTTNBP2 is important in neuroscience research because it regulates the density and size of dendritic spines, which are the primary sites of excitatory synapses in mammalian brains . Knockdown of CTTNBP2 in neurons reduces the density and size of dendritic spines and decreases the frequencies of miniature EPSCs, indicating its importance in synaptic function . Additionally, CTTNBP2 has been associated with autism spectrum disorder, making it a relevant target for research on neurodevelopmental disorders .

How do CTTNBP2 and CTTNBP2NL differ in their expression patterns?

CTTNBP2 and CTTNBP2NL exhibit distinctly different expression patterns:

• CTTNBP2 is predominantly expressed in the brain and shows neuron-specific expression .

• CTTNBP2NL is expressed at very low levels in the brain (approximately 20-25 fold lower than CTTNBP2) but shows more prominent expression in non-neuronal tissues such as skin, lungs, and spleen .

Quantitative RT-PCR analysis has confirmed that CTTNBP2NL mRNA levels are significantly lower than CTTNBP2 in various brain regions, including the cerebral cortex, cerebellum, hippocampus, and striatum . This expression pattern difference suggests that CTTNBP2, not CTTNBP2NL, is the primary cortactin-binding protein variant involved in neuronal functions .

What are the known splice variants of CTTNBP2 and how can they be identified?

Mouse CTTNBP2 has three identified splice variants:

• Short form - The dominant form expressed in the brain

• Long form

• Intron form

These variants can be identified using RT-PCR with specific oligonucleotide primers designed to discriminate between the different splicing forms. The primers used in previous research include:

• Primer A: 5′-CCTCCCTCTACTTTGCCACA-3′

• Primer B: 5′-GCCATCTTCGCAGGAGTAAT-3′

• Primer C: 5′-AAGAAATGAGGAAGTGGGTGAA-3′

When designing experiments to study CTTNBP2, researchers should be aware of these variants and consider which form they are targeting with their antibodies or other molecular tools.

How are CTTNBP2-specific antibodies typically generated?

CTTNBP2 polyclonal antibodies have been successfully generated through the following methodology:

• Immunizing rabbits with GST-CTTNBP2 (amino acids 498–625) recombinant protein

• Purifying the antibody through a GST-coupled affinity column to remove GST-specific antibodies

• Further purifying specific antibody using a GST-CTTNBP2 (amino acids 498–625)-conjugated column

This approach yields antibodies that are specific to CTTNBP2 and can be used for various applications including immunoblotting, immunoprecipitation, and immunofluorescence staining.

How can I ensure my CTTNBP2 antibody does not cross-react with CTTNBP2NL?

To ensure specificity of CTTNBP2 antibodies and prevent cross-reactivity with CTTNBP2NL:

• Target epitopes that are unique to CTTNBP2 and not present in CTTNBP2NL. Sequence comparison and alignment between the two proteins can identify CTTNBP2-specific regions.

• Perform validation experiments using cells that express either CTTNBP2 or CTTNBP2NL. For example, transfect cells with tagged versions of each protein and confirm that your antibody only detects the target protein.

• Include appropriate controls in your experiments, such as CTTNBP2 knockout/knockdown samples.

• Use immunoblotting to confirm the specificity of the antibody by checking that it recognizes proteins of the expected molecular weight in tissues where CTTNBP2 is known to be expressed (brain) but not in tissues where CTTNBP2NL is predominantly expressed (skin) .

When generating CTTNBP2NL-specific antibodies, researchers have used synthetic peptides corresponding to CTTNBP2NL-specific sequences as immunogens to minimize cross-reactivity with CTTNBP2 . A similar approach can be used for CTTNBP2-specific antibodies.

What validation steps should be performed for a newly generated CTTNBP2 antibody?

A comprehensive validation protocol for CTTNBP2 antibodies should include:

• Specificity testing:

  • Immunoblotting with recombinant CTTNBP2 protein

  • Testing in brain tissue (where CTTNBP2 is expressed) versus non-neuronal tissues

  • Testing in cells transfected with CTTNBP2 versus control cells

  • Testing against CTTNBP2NL to confirm lack of cross-reactivity

• Sensitivity assessment:

  • Titration experiments to determine optimal antibody concentration

  • Detection limit determination using serial dilutions of target protein

• Application-specific validation:

  • For immunohistochemistry: Confirm appropriate subcellular localization (dendritic spines)

  • For immunoprecipitation: Verify ability to pull down CTTNBP2 and its known interacting partners (cortactin)

  • For Western blotting: Confirm detection of expected molecular weight bands

• Knockout/knockdown validation:

  • Test antibody in samples where CTTNBP2 has been knocked down using miRNA (as described in the literature)

  • Confirm signal reduction or elimination in these samples

• Reproducibility testing:

  • Verify consistent results across multiple lots/batches of the antibody

  • Test in multiple experimental settings and by different researchers

What are the most effective methods for studying CTTNBP2 localization in neurons?

For studying CTTNBP2 localization in neurons, the following methodologies have proven effective:

• Immunofluorescence staining in cultured hippocampal neurons:

  • Use specific CTTNBP2 antibodies along with markers for dendritic spines (such as phalloidin for F-actin)

  • Co-stain with PSD-95 to mark postsynaptic density

  • Co-stain with cortactin to observe colocalization

• Live-cell imaging with GFP-tagged CTTNBP2:

  • Transfect neurons with GFP-CTTNBP2 constructs

  • Use time-lapse imaging to track CTTNBP2 dynamics

  • Combine with fluorescence recovery after photobleaching (FRAP) to assess mobility

• Super-resolution microscopy:

  • Techniques such as STORM or STED can provide higher-resolution images of CTTNBP2 localization within dendritic spines

• Electron microscopy with immunogold labeling:

  • For ultrastructural localization of CTTNBP2 at synapses

• In vivo imaging in brain slices:

  • To confirm that the localization observed in cultured neurons reflects the in vivo situation

Research has shown that CTTNBP2 is highly concentrated at dendritic spines in both cultured neurons and in the mouse brain, and it stably resides at these spines even after glutamate stimulation .

How can I effectively use CTTNBP2 antibodies for immunoprecipitation studies?

For effective immunoprecipitation (IP) of CTTNBP2 and its interacting partners:

• Sample preparation:

  • Use fresh brain tissue or cultured neurons

  • Lyse cells in a buffer that preserves protein-protein interactions (typically containing non-ionic detergents like Triton X-100 or NP-40)

  • Include protease inhibitors to prevent protein degradation

• Pre-clearing step:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Immunoprecipitation:

  • Incubate pre-cleared lysates with CTTNBP2 antibody (optimal concentration determined during validation)

  • Add protein A/G beads to capture antibody-protein complexes

  • Wash thoroughly to remove non-specifically bound proteins

• Detection of interacting partners:

  • Analyze immunoprecipitates by Western blotting for known or suspected interacting partners

  • Proven interactions that can be detected include cortactin, striatin, zinedin, and phocein

• Controls:

  • Include a negative control IP using non-specific IgG

  • Consider using CTTNBP2 knockdown samples as additional controls

This approach has successfully demonstrated interactions between CTTNBP2 and cortactin, as well as with striatin and zinedin (regulatory B subunits of protein phosphatase 2A) .

What are the recommended fixation and permeabilization methods for CTTNBP2 immunostaining?

For optimal CTTNBP2 immunostaining results in neuronal cultures:

• Fixation:

  • 4% paraformaldehyde in PBS for 15-20 minutes at room temperature

  • Alternatively, methanol fixation at -20°C for 10 minutes may be used for certain applications

• Permeabilization:

  • 0.1-0.2% Triton X-100 in PBS for 5-10 minutes

  • For some applications, 0.1% saponin may provide gentler permeabilization

• Blocking:

  • 3-5% BSA or normal serum (from the species in which the secondary antibody was raised)

  • Include 0.1% Triton X-100 in blocking buffer to maintain permeabilization

• Primary antibody incubation:

  • Dilute CTTNBP2 antibody in blocking buffer

  • Incubate overnight at 4°C for optimal results

• Secondary antibody incubation:

  • Use fluorophore-conjugated secondary antibodies

  • Incubate for 1-2 hours at room temperature

  • Include nuclear counterstain such as DAPI

• Mounting:

  • Use anti-fade mounting medium to preserve fluorescence

These protocols have been successfully employed to visualize CTTNBP2 at dendritic spines in cultured hippocampal neurons .

How does CTTNBP2 knockdown affect dendritic spine morphology and what controls should be included?

CTTNBP2 knockdown significantly impacts dendritic spine morphology, and proper experimental design requires careful controls:

Effects of CTTNBP2 knockdown:

• Reduced density of dendritic spines

• Decreased size of dendritic spines

• Lower frequencies of miniature EPSCs, consistent with reduced spine numbers

Recommended controls and experimental design:

• Control miRNA:

  • Use non-targeting miRNA constructs (e.g., control miRNA) expressed at similar levels to the CTTNBP2 miRNA

• Rescue experiments:

  • Include conditions where CTTNBP2 is re-expressed using a miRNA-resistant construct (BP2-rescue)

  • This confirms that observed effects are specific to CTTNBP2 loss rather than off-target effects

• Downstream partner overexpression:

  • Test whether overexpression of downstream partners (e.g., cortactin) can rescue the knockdown phenotype

  • This helps establish the hierarchical relationship between proteins in the pathway

• Time course analysis:

  • Monitor spine changes over time to distinguish between formation and maintenance defects

• Quantification methods:

  • Count spine density (number of spines per unit dendrite length)

  • Measure spine head width and length

  • Classify spines morphologically (mushroom, thin, stubby)

  • Evaluate spine dynamics using time-lapse imaging

Research has shown that the defects caused by CTTNBP2 knockdown can be rescued by overexpression of cortactin but not by expression of a CTTNBP2 mutant protein lacking the cortactin interaction , indicating that cortactin acts downstream of CTTNBP2 in spinogenesis.

How can I investigate the interaction between CTTNBP2 and the striatin/PP2A complex?

To investigate the interaction between CTTNBP2 and the striatin/PP2A complex:

• Coimmunoprecipitation studies:

  • Immunoprecipitate CTTNBP2 from brain extracts and probe for striatin, zinedin, and phocein

  • Perform reciprocal IPs using antibodies against striatin family proteins

  • Include appropriate controls (IgG control, CTTNBP2 knockdown)

• Domain mapping experiments:

  • Generate domain deletion constructs of CTTNBP2 to identify regions required for striatin interaction

  • The N-terminal region and coiled-coil domain (NC domain) of CTTNBP2 have been shown to be sufficient for interaction with striatin

  • Similarly, the N-terminal coiled-coil (N1) domain of striatin is involved in association with CTTNBP2

• Subcellular localization studies:

  • Perform immunofluorescence staining to examine colocalization of CTTNBP2 and striatin/zinedin in dendritic spines

  • Use CTTNBP2 knockdown to assess whether striatin/zinedin localization to spines depends on CTTNBP2

• Functional studies:

  • Investigate whether CTTNBP2 affects the phosphatase activity of the PP2A complex

  • Identify potential substrates of the CTTNBP2-striatin-PP2A complex in dendritic spines

• Line scan analysis:

  • Measure the immunoreactivities of striatin and zinedin in dendritic spines in control versus CTTNBP2 knockdown neurons

  • Research has shown that striatin and zinedin signals in control spines are at least twofold higher than in CTTNBP2-knockdown spines

Research has demonstrated that CTTNBP2 is required for the synaptic distribution of striatin and zinedin, suggesting that CTTNBP2 targets the PP2A complex to dendritic spines .

What experimental approaches can distinguish between the roles of CTTNBP2 and CTTNBP2NL?

To differentiate between the functions of CTTNBP2 and CTTNBP2NL, consider these experimental approaches:

• Expression analysis:

  • Compare expression levels in different tissues using Western blotting and quantitative RT-PCR

  • Research has shown that CTTNBP2 is predominantly expressed in brain, while CTTNBP2NL shows higher expression in non-neuronal tissues

• Subcellular localization:

  • Examine the distribution patterns of both proteins when expressed in the same cell type

  • In COS cells, CTTNBP2 colocalizes with cortactin at the cell cortex and intracellular puncta, while CTTNBP2NL associates with stress fibers

• Functional replacement studies:

  • Test whether CTTNBP2NL can rescue the phenotypes caused by CTTNBP2 knockdown in neurons

  • Research indicates that CTTNBP2NL does not show activity in the regulation of dendritic spinogenesis

• Protein interaction comparisons:

  • While both proteins interact with cortactin, they may have different effects on cortactin localization and function

  • Investigate whether both proteins interact with the same set of partners (e.g., striatin family proteins) and whether these interactions have similar functional consequences

• Cellular function assessment:

  • Use specific knockdown of each protein in appropriate cell types (neurons for CTTNBP2, non-neuronal cells for CTTNBP2NL)

  • Evaluate the impact on cell-type specific functions

These approaches can help delineate the distinct roles of these homologous proteins in different cellular contexts and determine whether they have evolved specialized functions in different tissues.

How should I interpret conflicting results when using different CTTNBP2 antibodies?

When encountering conflicting results with different CTTNBP2 antibodies, consider these analytical approaches:

• Epitope differences:

  • Determine the epitopes recognized by each antibody

  • Antibodies targeting different regions may detect different splice variants or post-translationally modified forms

  • Some epitopes may be masked in certain protein complexes or conformations

• Validation status:

  • Review the validation data for each antibody

  • Well-validated antibodies with confirmed specificity should be given more weight

  • Check whether antibodies have been validated in your specific application (Western blot, IP, IHC)

• Confirmatory approaches:

  • Use alternative methods to verify results (e.g., RNA interference, overexpression)

  • If possible, test with knockout/knockdown controls for each antibody

  • Consider using tagged CTTNBP2 constructs and detecting with anti-tag antibodies

• Reconciliation strategies:

  • Determine if differences reflect biology rather than technical issues

  • Consider whether antibodies might be detecting different CTTNBP2 splice variants (short, long, or intron forms)

  • Assess whether post-translational modifications affect antibody recognition

• Experimental conditions:

  • Standardize fixation, permeabilization, and staining protocols

  • Test different blocking conditions to reduce non-specific binding

  • Optimize antibody concentrations for each application

The biological relevance of your findings should be confirmed using functional approaches, such as CTTNBP2 knockdown and rescue experiments .

What are the common pitfalls when using CTTNBP2 antibodies for quantitative analyses?

When using CTTNBP2 antibodies for quantitative analyses, researchers should be aware of these common pitfalls:

• Antibody saturation:

  • Using too high concentrations can lead to non-specific binding and false positives

  • Establish a standard curve to ensure measurements are within the linear range

• Inconsistent sample preparation:

  • Variations in fixation time or conditions can affect epitope accessibility

  • Standardize all preparation steps, including lysis buffer composition, incubation times, and temperatures

• Reference protein selection:

  • When normalizing Western blot data, choosing appropriate loading controls is crucial

  • Consider using neuron-specific references when analyzing brain samples

• Background subtraction methods:

  • Different approaches to background correction can significantly impact quantitative results

  • Document and consistently apply your background subtraction methodology

• Splice variant considerations:

  • Ensure your quantification accounts for all relevant CTTNBP2 splice variants

  • Antibodies may have different affinities for different variants

• Image acquisition parameters:

  • For immunofluorescence quantification, consistent exposure settings are essential

  • Avoid saturated pixels which prevent accurate quantification

• Dendritic spine analysis specifics:

  • When quantifying CTTNBP2 at dendritic spines, consistent criteria for spine identification are necessary

  • Use line scan analysis to measure relative distributions across spine and shaft, as demonstrated in published research

Researchers should include appropriate positive and negative controls and validate their quantification methods against established standards in the field.

How can I troubleshoot weak or absent CTTNBP2 immunostaining signal in brain tissue?

If experiencing weak or absent CTTNBP2 immunostaining signal in brain tissue, consider these troubleshooting steps:

• Tissue fixation optimization:

  • Test different fixation methods (paraformaldehyde concentrations, post-fixation times)

  • Consider perfusion fixation for better preservation of brain tissue

  • Try antigen retrieval methods to unmask epitopes (heat-induced or enzymatic)

• Antibody-specific considerations:

  • Increase antibody concentration or incubation time

  • Try different CTTNBP2 antibodies targeting different epitopes

  • Use signal amplification systems (TSA, ABC method)

• Permeabilization adjustments:

  • Test different detergent types and concentrations

  • Extend permeabilization time to improve antibody penetration in thick sections

• Blocking optimization:

  • Try different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time to reduce background

• Technical validation:

  • Confirm antibody functionality using positive control tissues (cortex, hippocampus)

  • Include a positive control protein known to be expressed in the same region

  • Verify primary antibody activity with a dot blot or Western blot

• Age and brain region considerations:

  • Check whether CTTNBP2 expression varies with age or brain region

  • Ensure you're examining regions with known CTTNBP2 expression

• Detection system optimization:

  • Use a more sensitive secondary antibody or detection system

  • Try fluorophores with different excitation/emission spectra

  • Adjust microscope settings (exposure time, gain, etc.)

When troubleshooting, methodically change one variable at a time and document all modifications to identify the critical factors affecting your immunostaining results.

How might CTTNBP2 antibodies be used to investigate its role in autism spectrum disorders?

CTTNBP2 antibodies can be valuable tools for investigating the protein's role in autism spectrum disorders (ASD) through several research approaches:

• Expression studies in ASD models:

  • Compare CTTNBP2 expression levels and patterns in postmortem brain tissue from individuals with ASD versus neurotypical controls

  • Examine CTTNBP2 expression in animal models of ASD using immunohistochemistry and Western blotting

• Mutation-specific antibodies:

  • Develop antibodies that specifically recognize ASD-associated CTTNBP2 variants

  • Use these to study the expression and localization of mutant forms

• Protein interaction studies:

  • Investigate whether ASD-associated mutations alter CTTNBP2's interactions with partners like cortactin or striatin/PP2A

  • Perform coimmunoprecipitation experiments to identify altered protein complexes

• Spine morphology analyses:

  • Examine dendritic spine abnormalities in ASD models and correlate with CTTNBP2 distribution

  • Investigate whether spine morphology defects in ASD can be rescued by modulating CTTNBP2 expression

• Circuit-level investigations:

  • Use CTTNBP2 antibodies to identify specific neuronal populations affected in ASD

  • Combine with electrophysiology to correlate CTTNBP2 expression with functional changes

• Developmental studies:

  • Track CTTNBP2 expression during critical periods of brain development

  • Determine whether developmental trajectories of CTTNBP2 expression differ in ASD models

Since CTTNBP2 regulates dendritic spine formation and has been associated with ASD , studying its expression and function in autism models may provide insights into the synaptic basis of this neurodevelopmental disorder.

What strategies can be employed to develop phospho-specific CTTNBP2 antibodies?

Developing phospho-specific CTTNBP2 antibodies requires careful planning and validation:

• Phosphorylation site identification:

  • Use phosphoproteomic approaches to identify physiologically relevant phosphorylation sites

  • Analyze CTTNBP2 sequence for consensus phosphorylation motifs recognized by known kinases

  • Consider sites that might regulate interaction with binding partners like cortactin or striatin

• Peptide design for immunization:

  • Design phosphopeptides (10-15 amino acids) containing the phosphorylated residue in the center

  • Ensure the sequence is unique to CTTNBP2 and not present in CTTNBP2NL

  • Consider coupling to a carrier protein (KLH or BSA) to enhance immunogenicity

• Immunization and antibody production:

  • Immunize rabbits or other suitable host animals with the phosphopeptide

  • Monitor antibody titers using ELISA against phosphorylated and non-phosphorylated peptides

• Purification strategies:

  • Employ a two-step affinity purification process:
    a) First, purify against the phosphopeptide column
    b) Then, negatively select using a non-phosphorylated peptide column

  • This removes antibodies that recognize the non-phosphorylated form

• Rigorous validation:

  • Test specificity using Western blotting of samples treated with phosphatases

  • Confirm with samples from cells treated with kinase activators/inhibitors

  • Verify using CTTNBP2 constructs with phospho-mimetic and phospho-dead mutations

• Functional validation:

  • Determine whether phosphorylation status changes with neuronal activity

  • Investigate whether phosphorylation affects CTTNBP2 localization to dendritic spines

  • Assess whether phosphorylation alters interaction with binding partners

Phospho-specific antibodies would be valuable for understanding how CTTNBP2 function is regulated by post-translational modifications during neuronal development and in response to synaptic activity.

How can advanced imaging techniques be combined with CTTNBP2 antibodies to better understand its dynamic regulation?

Combining advanced imaging techniques with CTTNBP2 antibodies can provide deeper insights into its dynamic regulation:

• Super-resolution microscopy:

  • Techniques like STORM, PALM, or STED can resolve CTTNBP2 localization within dendritic spines at nanoscale resolution

  • Use multi-color super-resolution to examine CTTNBP2 positioning relative to synaptic proteins

  • This can reveal previously undetectable subsynaptic compartmentalization

• Live-cell single-molecule tracking:

  • Use antibody fragments (Fab) conjugated to quantum dots or organic dyes

  • Track movement of individual CTTNBP2 molecules in living neurons

  • Analyze diffusion coefficients in different cellular compartments

• FRET/FLIM approaches:

  • Develop FRET pairs with CTTNBP2 and its binding partners (cortactin, striatin)

  • Monitor protein-protein interactions in real-time during synaptic activity

  • Use FLIM to obtain quantitative measurements of molecular proximity

• Optogenetic manipulation combined with imaging:

  • Light-activate signaling pathways that might regulate CTTNBP2

  • Simultaneously monitor CTTNBP2 redistribution using immunofluorescence

  • This can reveal cause-effect relationships in CTTNBP2 regulation

• Expansion microscopy:

  • Physically expand preserved tissue to increase effective resolution

  • Combine with CTTNBP2 immunostaining for improved visualization of nanoscale features

  • Particularly useful for mapping CTTNBP2 distribution in intact brain tissue

• Correlative light and electron microscopy (CLEM):

  • Immunolabel CTTNBP2 for fluorescence imaging

  • Process the same sample for electron microscopy

  • Correlate CTTNBP2 localization with ultrastructural features

• Fluorescence recovery after photobleaching (FRAP):

  • FRAP has already been used to demonstrate that CTTNBP2 modulates the mobility of cortactin in neurons

  • This technique can be further employed to investigate how CTTNBP2 mobility is itself regulated by neuronal activity or signaling pathways