ERC2 Antibody

What is ERC2 and what are its key functions in neural tissues?

ERC2, also known as CAST1, is a CAZ protein involved in organizing the cytomatrix at nerve terminal active zones that regulates neurotransmitter release. It interacts with the conserved RIM PDZ domain via an unusual PDZ binding motif . ERC2 is thought to act together with Bassoon (BSN) to regulate neurotransmitter release and may recruit liprin-alpha proteins to the CAZ . The protein functions as part of a complex network involved in presynaptic organization, potentially linking Rab6-mediated membrane trafficking at the Golgi complex to neuronal membrane traffic at the active zone via RIMs .

What species reactivity can be expected with ERC2 antibodies?

Based on current available antibodies, species reactivity varies by manufacturer and clone:

When selecting an antibody, verify species cross-reactivity with the manufacturer, as some antibodies may have limited species coverage.

What applications are ERC2 antibodies optimized for?

ERC2 antibodies are validated for several applications with varying dilution requirements:

For optimal results, titration is recommended for each specific experimental system as results can be tissue and preparation-dependent .

How should ERC2 antibodies be stored and handled for maximum stability?

For lyophilized antibodies (e.g., Synaptic Systems), reconstitute by adding 50 μl H₂O to achieve a 1mg/ml solution in PBS. After reconstitution, aliquot and store at -20°C to -80°C. Importantly, antibodies should be stored at +4°C when still lyophilized, and freezing of the lyophilized product should be avoided .

For liquid formulations (e.g., Proteintech), store at -20°C in the buffer provided (typically PBS with 0.02% sodium azide and 50% glycerol at pH 7.3). These formulations are generally stable for one year after shipment, and for the 20μl size which contains 0.1% BSA, aliquoting is unnecessary for -20°C storage .

What is the best approach to validate ERC2 antibody specificity?

Knockout (KO) validation is the gold standard for antibody specificity. The Synaptic Systems ERC2 antibody (143 103) has been KO-validated as referenced in PubMed: 27422015 . For antibodies without KO validation, consider these approaches:

• Western blot analysis showing a single band at the expected molecular weight (111 kDa calculated, 115-120 kDa observed)

• Comparative analysis with siRNA knockdown samples

• Testing in tissues known to express ERC2 (brain tissue shows high expression) versus non-expressing tissues

• Blocking peptide controls using the immunogen peptide (e.g., synthetic peptide corresponding to AA 655 to 670 from rat ERC2)

How can I optimize ERC2 detection in immunohistochemistry applications?

For optimal IHC results with ERC2 antibodies:

• Antigen retrieval: Use TE buffer pH 9.0 as suggested for the Proteintech antibody. Alternatively, citrate buffer pH 6.0 may be used .

• Fixation: 4% paraformaldehyde fixation is typically suitable

• Sectioning: 5-10 μm sections are recommended for paraffin-embedded tissues

• Blocking: Use 5-10% normal serum from the same species as the secondary antibody

• Primary antibody dilution: Begin with 1:200-1:800 as recommended

• Controls: Include a negative control (no primary antibody) and positive control (brain tissue)

How can I distinguish between ERC1 and ERC2 in experimental systems?

Distinguishing between ERC1 and ERC2 can be challenging as they share an identical C-terminal sequence . Consider these approaches:

• Use antibodies targeting non-conserved regions: Select antibodies raised against unique regions of ERC2. For example, the Synaptic Systems antibody targets amino acids 655-670 of rat ERC2 .

• Expression pattern analysis: ERC2 (CAST1) is expressed only as a single RIM binding variant, whereas ERC1 has two splice variants (1a and 1b). ERC1b (CAST2a) is brain-specific, while ERC1a is ubiquitously expressed .

• Subcellular fractionation: ERC2 is predominantly found in crude synaptosomal (P2) fractions, while ERC1b has a more diffuse distribution including cytosolic (S3) and synaptosomal cytosol (LS2) fractions .

• Developmental expression: Monitor temporal expression patterns, as both proteins show increasing expression during postnatal development, but with different profiles .

What are the critical protein-protein interactions of ERC2 that can be studied using antibodies?

ERC2 participates in several important protein interactions that can be studied using co-immunoprecipitation and other antibody-based techniques:

• RIM interaction: ERC2 interacts with the PDZ domain of RIM through its C-terminal PDZ-binding motif. This interaction is crucial for active zone organization .

• Syntenin-1 interaction: ERC2 binds syntenin-1 through its C-terminal PDZ-binding motif, with PDZ2 of syntenin-1 being particularly important. This interaction influences presynaptic clustering .

• ERC2 multimerization: ERC2 self-associates through regions between amino acids 693-957, which is important for its function. Co-IP experiments with differentially tagged ERC2 variants can study this multimerization .

• Bassoon/Piccolo interactions: ERC2 interacts with these scaffolding proteins, and these interactions regulate neurotransmitter release .

• Liprin-α interactions: ERC2 may recruit liprin-α proteins to the CAZ .

How can ERC2 antibodies be used to study presynaptic development and plasticity?

ERC2 antibodies can be valuable tools for investigating presynaptic development and plasticity through multiple approaches:

• Developmental time-course: Immunoblot analysis reveals that ERC2 expression increases during postnatal development, suggesting its role in synapse maturation . Using ERC2 antibodies to track expression levels at different developmental stages can provide insights into synaptogenesis.

• Activity-dependent changes: Immunofluorescence imaging after various activity manipulations (tetanic stimulation, TTX blockade, etc.) can reveal changes in ERC2 localization or expression.

• Presynaptic assembly: ERC2 promotes syntenin-1 clustering at presynaptic sites through both PDZ interaction and ERC multimerization. When expressed in neurons, fragments of ERC2 (amino acids 1-957) cause significant clustering of syntenin-1 at presynaptic sites (82.3 ± 4.3% of ERC2 clusters were syntenin-1 positive), while ERC2 fragments lacking multimerization capabilities (amino acids 1-693) failed to promote syntenin-1 clustering .

• Colocalization studies: Double-immunostaining with ERC2 antibodies and other presynaptic markers can map the temporal sequence of CAZ assembly.

What are the most common issues when using ERC2 antibodies and how can they be resolved?

Several common issues may arise when working with ERC2 antibodies:

• High background in immunostaining:

  • Increase blocking time and concentration

  • Reduce primary antibody concentration

  • Include additional washing steps

  • Use tissue from ERC2 knockout animals as negative controls

• Multiple bands in Western blot:

  • Optimize sample preparation (consider phosphatase inhibitors)

  • Use fresh tissue samples, particularly for brain tissue

  • Ensure complete transfer of high molecular weight proteins

  • Validate with knockout controls when possible

• Weak or no signal:

  • For brain tissues, ensure proper region selection as ERC2 expression varies across brain regions

  • Verify antibody storage conditions haven't compromised activity

  • For IHC, optimize antigen retrieval methods (try both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Consider detergent concentration in lysis buffers for WB applications

How can I optimize double immunofluorescence staining with ERC2 and other presynaptic markers?

For successful double immunostaining with ERC2 and other presynaptic proteins:

• Sequential staining approach:

  • Complete staining with the first primary and secondary antibodies

  • Apply additional blocking step to prevent cross-reactivity

  • Proceed with the second set of antibodies

• Antibody selection considerations:

  • Choose primary antibodies from different host species

  • Verify secondary antibodies do not cross-react

  • Select fluorophores with minimal spectral overlap

  • Consider using directly conjugated antibodies for one marker

• Controls:

  • Single staining controls for each antibody

  • Secondary antibody-only controls

  • Absorption controls with blocking peptides

• Sample preparation optimization:

  • Test different fixation protocols (duration, temperature)

  • Optimize permeabilization conditions

  • Consider using Triton X-100 at 0.1-0.3% for membrane proteins

What considerations should be made when studying ERC2 in different neural preparations?

Different neural preparations require specific considerations when studying ERC2:

• Primary neuronal cultures:

  • Allow sufficient time for synapse formation (typically >10 DIV)

  • Verify neuronal health and density

  • Consider co-culture with glial cells for optimal synapse development

• Brain slices:

  • Optimize fixation time to preserve antigenicity while ensuring tissue penetration

  • Section thickness affects antibody penetration (30-50 μm typically works well)

  • Extended washing may be necessary for thick sections

• Electron microscopy immunogold labeling:

  • Mild fixation conditions to preserve antigenicity

  • Careful consideration of embedding media

  • May require specialized secondary antibodies conjugated to gold particles

• Synaptosome preparations:

  • ERC2 is primarily found in crude synaptosomal (P2) fractions

  • Careful homogenization to preserve presynaptic structures

  • Fresh preparation is recommended for optimal results

How is ERC2 research contributing to our understanding of synaptic disorders?

ERC2's critical role in organizing the presynaptic active zone suggests its potential involvement in neurological and psychiatric disorders characterized by synaptic dysfunction. Research using ERC2 antibodies can help:

• Characterize changes in ERC2 expression or localization in animal models of neurological disorders

• Identify alterations in protein interactions between ERC2 and its binding partners in disease states

• Assess whether therapeutic interventions normalize ERC2-dependent synaptic organization

• Investigate the role of ERC2 in synapse formation and maintenance, processes often disrupted in neurodevelopmental disorders

This research direction holds promise for understanding conditions such as autism spectrum disorders, schizophrenia, and neurodegenerative diseases where synaptic dysfunction is implicated.

What emerging techniques can be combined with ERC2 antibodies for advanced synaptic research?

Several cutting-edge techniques can enhance ERC2 research:

• Super-resolution microscopy (STORM, STED, PALM):

  • Overcomes the diffraction limit to visualize nanoscale organization of ERC2 at the active zone

  • Can resolve the precise spatial relationship between ERC2 and other CAZ proteins

• Proximity labeling techniques (BioID, APEX):

  • Identify proteins in close proximity to ERC2 in living cells

  • Discover novel ERC2 interacting partners at the active zone

• Live imaging with genetically encoded tags:

  • Monitor dynamics of ERC2 during synapse formation and plasticity

  • Requires careful validation with antibodies to ensure tagged proteins behave like endogenous proteins

• Mass spectrometry following immunoprecipitation:

  • Identify post-translational modifications of ERC2

  • Characterize composition of ERC2-containing protein complexes under different conditions

• CRISPR-Cas9 genome editing:

  • Generate tagged endogenous ERC2 for improved specificity

  • Create specific mutations to study structure-function relationships

These approaches, combined with validated ERC2 antibodies, can significantly advance our understanding of presynaptic organization and function.