STXBP1 Antibody, Biotin conjugated

What is STXBP1 and what is its role in neuronal function?

STXBP1 (Syntaxin Binding Protein 1), also known as MUNC18-1, is a critical protein involved in the docking and fusion of synaptic vesicles, which is essential for neurotransmitter release into the synapse. STXBP1 binds syntaxin (a component of the synaptic vesicle fusion machinery) in a 1:1 ratio and can interact with syntaxins 1, 2, and 3, but not syntaxin 4 . Upon assembly with Syntaxin 1 and other SNARE proteins, STXBP1 forms a tight complex that drives the fusion of synaptic vesicles with the presynaptic plasma membrane, facilitating neurotransmitter exocytosis .

STXBP1 has significant clinical relevance as STXBP1 haploinsufficiency represents one of the most common genetic causes of developmental and epileptic encephalopathies . Mouse models with STXBP1 haploinsufficiency (Stxbp1+/-) demonstrate seizure phenotypes characterized by myoclonic jerks and spike-wave discharges, providing valuable insights into the human condition .

What applications are Biotin-conjugated STXBP1 antibodies validated for?

Biotin-conjugated STXBP1 antibodies have been validated for multiple research applications based on standardized experimental protocols. The validation data indicates:

The antibodies have been characterized using standardized experimental protocols based on comparing read-outs in knockout cell lines and isogenic parental controls, enhancing confidence in their specificity and performance .

What is the recommended protocol for antigen retrieval when using Biotin-conjugated STXBP1 antibodies in immunohistochemistry?

For optimal results in immunohistochemistry applications with Biotin-conjugated STXBP1 antibodies, the following antigen retrieval protocols are recommended:

• Primary method: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

These recommendations apply to both the polyclonal (Biotin-11459) and monoclonal (Biotin-67137) antibodies when working with formalin-fixed paraffin-embedded tissues such as mouse brain samples . The choice between these two methods may depend on the specific tissue preparation and fixation conditions. Researchers should validate both methods with their specific samples to determine which produces optimal signal-to-noise ratio.

What are the proper storage conditions for maintaining Biotin-conjugated STXBP1 antibody activity?

To maintain optimal activity of Biotin-conjugated STXBP1 antibodies, the following storage conditions are recommended:

• Store at -20°C

• Avoid exposure to light (due to the biotin conjugate)

• The antibodies are stable for one year after shipment when stored properly

• The storage buffer consists of PBS with 50% Glycerol, 0.05% Proclin300, 0.5% BSA, pH 7.3

• Aliquoting is unnecessary for -20°C storage

Adhering to these storage recommendations will help ensure the antibodies maintain their specificity and activity for the expected shelf life.

What is the expected molecular weight of STXBP1 in Western blot applications?

When using Biotin-conjugated STXBP1 antibodies in Western blot applications, researchers should expect to observe the following:

• Calculated molecular weight: 68 kDa (based on the 594 amino acid sequence)

• Observed molecular weight: 65-68 kDa range

This information is consistent across product data sheets and validation studies . The slight variation in observed molecular weight may result from post-translational modifications or differences in electrophoresis conditions. When analyzing Western blot results, researchers should focus on bands appearing within this molecular weight range.

How do monoclonal and polyclonal Biotin-conjugated STXBP1 antibodies differ in their research applications?

The choice between monoclonal (Biotin-67137) and polyclonal (Biotin-11459) STXBP1 antibodies should be based on the specific research requirements:

Monoclonal antibody (Biotin-67137):

• Clone: 1B5B3

• Isotype: IgG2a

• Primarily validated for IHC applications

• Demonstrates higher specificity for a single epitope

• May provide more consistent lot-to-lot reproducibility

• Particularly useful for comparative studies requiring precise epitope recognition

Polyclonal antibody (Biotin-11459):

• Host: Rabbit / IgG

• Validated for both WB (1:500-1:2000) and IHC (1:50-1:500)

• Recognizes multiple epitopes on the STXBP1 protein

• May provide stronger signal amplification

• Potentially more resistant to antigen changes due to fixation or denaturation

Researchers should select the appropriate antibody based on their specific application, with monoclonal antibodies generally preferred for high-specificity applications and polyclonal antibodies for maximum sensitivity or when antigen conformational changes may occur.

What experimental controls should be implemented when validating the specificity of Biotin-conjugated STXBP1 antibodies?

Rigorous validation of antibody specificity is essential for generating reliable research data. For Biotin-conjugated STXBP1 antibodies, the following controls should be implemented:

• Knockout validation: Compare antibody reactivity between STXBP1 knockout cell lines and isogenic parental controls to confirm specificity

• Positive tissue controls: Use known STXBP1-expressing tissues such as:

  • Mouse brain tissue (particularly cortical regions)

  • Y79 cells (for Western blot applications)

• Negative controls:

  • Primary antibody omission

  • Isotype controls to detect non-specific binding

  • Blocking peptide competition assays

• Signal verification:

  • Confirm expected molecular weight (65-68 kDa) in Western blot applications

  • Verify expected cellular localization pattern in IHC/IF (presynaptic localization)

• Cross-reactivity assessment:

  • Test antibody performance across multiple species when relevant (human, mouse, rat samples)

These comprehensive validation approaches align with current best practices in antibody validation as highlighted in collaborative initiatives addressing antibody reproducibility issues .

How can Biotin-conjugated STXBP1 antibodies be utilized in studying STXBP1-related epileptic encephalopathies?

Biotin-conjugated STXBP1 antibodies serve as valuable tools for investigating STXBP1-related epileptic encephalopathies through multiple research approaches:

• Mouse model characterization:

  • Detection of STXBP1 protein levels in heterozygous (Stxbp1+/-) mouse models that recapitulate human seizure phenotypes

  • Comparison of STXBP1 expression in different genetic backgrounds (e.g., C57BL/6J, 129Sv)

  • Analysis of selective STXBP1 haploinsufficiency in GABAergic neurons using conditional knockout models

• Protein stability assessment:

  • Evaluation of STXBP1 mutant protein stability using cycloheximide chase experiments in neuronal cultures

  • Comparison of wild-type versus disease-causing STXBP1 variants

• Brain region analysis:

  • IHC application to identify seizure foci through co-staining with activity markers like c-Fos

  • Mapping STXBP1 expression patterns in neocortical areas implicated in epileptogenesis

• Therapeutic evaluation:

  • Monitoring changes in STXBP1 expression levels following antiepileptic drug treatment (e.g., levetiracetam)

  • Assessing correlation between protein expression and seizure suppression

These applications enable researchers to investigate the molecular mechanisms underlying STXBP1 haploinsufficiency and explore potential therapeutic strategies for associated epileptic encephalopathies.

What are the key considerations for optimizing immunohistochemistry protocols with Biotin-conjugated STXBP1 antibodies?

Optimizing immunohistochemistry protocols with Biotin-conjugated STXBP1 antibodies requires attention to several critical parameters:

• Fixation considerations:

  • Formalin-fixed paraffin-embedded (FFPE) tissues require appropriate antigen retrieval

  • Fresh-frozen sections may provide better epitope preservation but require optimization of fixation time

• Antigen retrieval optimization:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • Optimization of retrieval duration and temperature based on tissue type

• Antibody dilution titration:

  • Starting dilution range: 1:50-1:500 for both polyclonal and monoclonal antibodies

  • Sample-dependent optimization recommended to determine optimal signal-to-noise ratio

• Blocking considerations:

  • Use appropriate blocking to minimize background from endogenous biotin

  • Consider using avidin/biotin blocking kit for tissues with high endogenous biotin (e.g., liver, kidney)

• Detection system selection:

  • Streptavidin-HRP systems for chromogenic detection

  • Fluorescent streptavidin conjugates for multiplexing applications

• Positive and negative controls:

  • Mouse brain tissue serves as a reliable positive control

  • Include secondary-only controls to assess background

These optimization steps will help researchers achieve specific and reproducible STXBP1 detection in tissue samples.

What approaches are recommended for troubleshooting non-specific binding when using Biotin-conjugated STXBP1 antibodies?

When encountering non-specific binding with Biotin-conjugated STXBP1 antibodies, researchers should systematically address potential issues:

• Endogenous biotin interference:

  • Implement avidin/biotin blocking steps before primary antibody incubation

  • Consider testing with non-biotinylated primary antibody versions if persistent biotin interference occurs

• Antibody concentration adjustment:

  • Further titrate antibody dilutions (starting from the recommended 1:50-1:500 range)

  • Optimize primary antibody incubation time and temperature

• Blocking protocol modification:

  • Test different blocking reagents (BSA, normal serum, commercial blockers)

  • Increase blocking concentration or duration

• Washing optimization:

  • Increase number and duration of wash steps

  • Consider adding low concentrations of detergent (0.05-0.1% Tween-20) to wash buffers

• Antigen retrieval assessment:

  • Compare recommended TE buffer (pH 9.0) versus citrate buffer (pH 6.0)

  • Adjust retrieval duration and temperature

• Secondary detection system:

  • If using streptavidin-based detection, ensure proper streptavidin dilution

  • Consider alternative detection systems if background persists

• Tissue-specific considerations:

  • For brain tissue with high lipid content, optimize permeabilization steps

  • Evaluate fixation methods and post-fixation storage conditions

Systematic implementation of these troubleshooting approaches should help resolve non-specific binding issues and improve signal specificity.

How can researchers quantify STXBP1 expression levels using Biotin-conjugated antibodies?

Accurate quantification of STXBP1 expression using Biotin-conjugated antibodies can be achieved through several methodological approaches:

• Western blot quantification:

  • Use the validated dilution range (1:500-1:2000) for Biotin-11459

  • Include recombinant STXBP1 standards for absolute quantification

  • Normalize to appropriate loading controls (beta-actin, GAPDH)

  • Employ digital image analysis software for densitometry measurements

• Immunohistochemistry quantification:

  • Standardize image acquisition parameters (exposure time, gain settings)

  • Implement automated image analysis for:

    • Cell counting (percent STXBP1-positive cells)

    • Mean fluorescence/staining intensity measurements

    • Area of STXBP1-positive staining

• Multi-parameter analysis:

  • Co-stain with neuronal markers (MAP2) or synaptic markers (Synapsin, VAMP2, Syntaxin-1)

  • Analyze co-localization coefficients

  • Evaluate synaptic density in relation to STXBP1 expression

• Comparing wild-type vs. mutant expression:

  • Use standardized protocols when analyzing disease-causing mutations

  • Normalize to wild-type controls when assessing protein stability

  • Consider cycloheximide chase experiments for protein half-life determination

These quantification approaches provide researchers with robust methods to assess STXBP1 expression levels across different experimental conditions and genotypes.

What are the considerations for multiplexing Biotin-conjugated STXBP1 antibodies with other markers in neuronal research?

Multiplexing Biotin-conjugated STXBP1 antibodies with other neuronal markers requires careful planning to achieve optimal results:

• Detection system compatibility:

  • Biotin-streptavidin systems limit additional biotin-based detection in the same sample

  • Consider fluorescent streptavidin conjugates (e.g., streptavidin-Alexa Fluor) for one channel

  • Use directly conjugated antibodies for additional markers

• Recommended neuronal marker combinations:

  • Presynaptic markers: Syntaxin-1, VAMP2, Synapsin

  • Neuronal cytoskeletal markers: MAP2

  • Activation markers: c-Fos (for seizure activity)

• Species compatibility:

  • Select additional primary antibodies raised in different host species than the STXBP1 antibody

  • For mouse monoclonal Biotin-STXBP1 (Biotin-67137), pair with rabbit, chicken, or goat primaries

  • For rabbit polyclonal Biotin-STXBP1 (Biotin-11459), pair with mouse, chicken, or goat primaries

• Sequential staining considerations:

  • When antibody species conflicts cannot be avoided, consider sequential staining protocols

  • Complete first primary-secondary staining cycle

  • Block with excess unconjugated secondary antibody

  • Proceed with second primary-secondary staining cycle

• Signal separation optimization:

  • Select fluorophores with minimal spectral overlap

  • Implement appropriate controls to assess bleed-through

  • Use spectral unmixing for closely overlapping signals

These considerations will enable researchers to effectively combine STXBP1 detection with other relevant markers in complex neuronal samples.

How do different fixation protocols affect the performance of Biotin-conjugated STXBP1 antibodies?

Fixation protocols significantly impact the performance of Biotin-conjugated STXBP1 antibodies, with several key considerations:

• Formalin fixation effects:

  • Standard 4% formaldehyde/paraformaldehyde fixation is compatible with both antibodies

  • Fixation duration affects epitope accessibility (typically 15-30 minutes for cultured neurons, 24 hours for tissue blocks)

  • Requires appropriate antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0)

• Alternative fixation methods:

  • Methanol fixation may improve detection of certain epitopes but can disrupt membrane structures

  • Glutaraldehyde should be avoided as it can cause high autofluorescence and excessive cross-linking

  • PFA/methanol combined protocols may provide balanced preservation of structure and epitope accessibility

• Fresh-frozen versus fixed tissues:

  • Fresh-frozen sections may preserve epitopes better but require optimization of post-sectioning fixation

  • FFPE tissues provide better morphological preservation but require more rigorous antigen retrieval

• Cell culture considerations:

  • For cultured neurons, 4% formaldehyde fixation for 15-20 minutes at room temperature is recommended

  • Permeabilization with 0.5% Triton X-100 improves antibody access to intracellular epitopes

• Post-fixation processing:

  • Prolonged storage of fixed tissues before processing may reduce antibody binding

  • Antigen retrieval parameters may need adjustment based on fixation-to-processing time

Researchers should validate their specific fixation protocol with proper controls to ensure optimal STXBP1 detection in their experimental system.

What are the key considerations for selecting between different Biotin-conjugated STXBP1 antibodies?

When selecting between different Biotin-conjugated STXBP1 antibodies, researchers should consider several factors to match the antibody properties with their specific experimental requirements:

• Application compatibility:

  • Polyclonal Biotin-11459: Validated for both WB (1:500-1:2000) and IHC (1:50-1:500)

  • Monoclonal Biotin-67137: Primarily validated for IHC (1:50-1:500)

• Species reactivity requirements:

  • Both antibodies: Human, mouse, rat reactivity

  • Biotin-67137: Additional pig reactivity

• Specificity versus sensitivity needs:

  • Monoclonal: Higher specificity for particular epitope, greater reproducibility

  • Polyclonal: Potentially higher sensitivity, recognition of multiple epitopes

• Experimental design considerations:

  • For precise localization studies: Consider monoclonal antibodies

  • For maximum signal detection: Consider polyclonal antibodies

  • For cross-species comparisons: Verify validated species reactivity

• Validation depth:

  • Review antibody characterization data using knockout controls

  • Assess published literature using specific catalog numbers

  • Consider preliminary testing with positive control samples

Careful consideration of these factors will help researchers select the most appropriate Biotin-conjugated STXBP1 antibody for their specific research needs, ultimately contributing to more reliable and reproducible experimental outcomes.

How does STXBP1 antibody research contribute to understanding neurological disorders?

Research utilizing STXBP1 antibodies has made significant contributions to understanding neurological disorders through several important avenues:

• Mechanistic insights into STXBP1-encephalopathy:

  • STXBP1 haploinsufficiency has been established as one of the most common genetic causes of developmental and epileptic encephalopathies

  • Antibody-based studies have revealed that cellular Munc18-1 (STXBP1) instability is the probable explanation for pathogenicity of STXBP1 mutations found in patients

• Mouse model validation:

  • Antibody studies have helped characterize Stxbp1+/- mouse models that recapitulate human seizure phenotypes

  • These models demonstrate construct, face, and predictive validity for studying STXBP1-encephalopathy

  • c-Fos and STXBP1 co-staining has implicated neocortical areas as seizure foci

• Cellular pathology characterization:

  • STXBP1 antibodies have enabled investigation of protein stability across different disease-causing mutations

  • Studies have demonstrated impaired cognitive performance, hyperactivity, and anxiety-like behavior in Stxbp1+/- mice

• Therapeutic development:

  • Antibody-based research has supported evaluation of antiepileptic drugs like levetiracetam in suppressing seizures in STXBP1-deficient models

  • These studies provide a foundation for targeted therapeutic approaches

• Technical advances:

  • Standardized antibody validation approaches have improved research tool reliability

  • Collaborative initiatives between academics, funders, and commercial antibody manufacturers are addressing reproducibility issues in STXBP1 research