SEZ6L2 Antibody, FITC conjugated

What is SEZ6L2 and why is it important in neuroscience research?

SEZ6L2 (Seizure Related 6 Homolog Like 2) is a type 1 transmembrane protein that is predominantly expressed in the brain. It belongs to the seizure-related gene 6 (SEZ6) family, which includes SEZ6, SEZ6L, and SEZ6L2. These family members have significant roles in influencing synapse numbers and dendritic morphology and are associated with various neurological and psychiatric disorders . Specifically, SEZ6L2 functions as part of the AMPA receptor complex, acting as a scaffolding protein that links GluR1 to adducin . Its importance in neuroscience research stems from its implications in synaptic function and potential role in pathological conditions, making it a valuable target for studying brain development, synaptic plasticity, and neurological disorders.

What are the key characteristics of SEZ6L2 antibodies?

SEZ6L2 antibodies are typically produced in rabbit hosts and are available in polyclonal formats . These antibodies target specific regions of the SEZ6L2 protein, such as the C-terminal region (amino acids 879-907) or other epitopes. The molecular weight of SEZ6L2 is calculated to be approximately 98 kDa (910 amino acids), but due to glycosylation, the observed molecular weight in experimental conditions is often around 150 kDa . SEZ6L2 antibodies are available in various conjugated forms, including FITC (fluorescein isothiocyanate), which is particularly useful for flow cytometry and immunofluorescence applications . These antibodies demonstrate reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across species .

What are the typical applications for SEZ6L2 antibodies in research?

SEZ6L2 antibodies are employed in various experimental techniques including:

• Western Blotting (WB): For detection and quantification of SEZ6L2 protein in tissue or cell lysates, with recommended dilutions typically ranging from 1:1000 to 1:4000 .

• Flow Cytometry (FACS): Particularly useful with FITC-conjugated antibodies, allowing for detection and sorting of cells expressing SEZ6L2 .

• ELISA: For quantitative measurement of SEZ6L2 in solution .

• Immunohistochemistry (IHC): For visualizing SEZ6L2 expression patterns in tissue sections .

These applications provide researchers with complementary approaches to investigate SEZ6L2 expression, localization, and function in various experimental contexts, from cellular to tissue levels.

What is the functional significance of SEZ6L2 in complement regulation and how can this be investigated using labeled antibodies?

Recent research has demonstrated that the SEZ6 family, with SEZ6L2 as a representative member, plays a significant role in complement regulation by specifically accelerating the dissociation of C3 convertases . This function positions SEZ6L2 as an important modulator of complement activation, a key component of innate immunity that can significantly impact neuroinflammatory processes. FITC-conjugated SEZ6L2 antibodies can be instrumental in investigating this function through flow cytometry to analyze the co-localization of SEZ6L2 with complement components on cell surfaces. Additionally, microscopy techniques using these fluorescent antibodies can visualize the spatial relationship between SEZ6L2 and complement regulators in tissue sections. To properly design such experiments, researchers should consider:

• Using appropriate controls, including isotype controls and cells lacking SEZ6L2 expression

• Implementing complement activation assays in conjunction with SEZ6L2 detection

• Analyzing co-localization through dual-labeling techniques

• Correlating SEZ6L2 expression levels with complement activation markers

This approach would provide insights into how SEZ6L2 participates in complement regulation in different cellular contexts and pathological conditions.

How does SEZ6L2 glycosylation affect antibody recognition, and what methodological approaches can address this challenge?

SEZ6L2 undergoes extensive glycosylation that can increase its apparent molecular weight from the calculated 98 kDa to approximately 150-170 kDa as observed in experimental conditions . This post-translational modification presents several challenges for antibody recognition:

• Epitope masking: Glycosylation can potentially obscure antibody binding sites

• Heterogeneous banding patterns: Variable glycosylation can result in multiple bands in Western blots

• Species and tissue-specific differences in glycosylation patterns

To address these challenges, researchers working with FITC-conjugated SEZ6L2 antibodies should consider the following methodological approaches:

• Deglycosylation treatments: Using enzymes like PNGase F to remove N-linked glycans before analysis

• Selection of antibodies targeting epitopes less likely to be affected by glycosylation

• Cross-validation using multiple antibodies targeting different epitopes

• Careful selection of positive controls with known glycosylation patterns

These approaches enable more accurate interpretation of results, particularly in comparative studies across different experimental models or clinical samples.

What is the relationship between SEZ6L2 and GluR1 in AMPA receptor function, and how can FITC-conjugated antibodies elucidate this interaction?

SEZ6L2 functions as part of the AMPA receptor complex, specifically acting as a scaffolding protein that links GluR1 to adducin . This interaction is critical for proper AMPA receptor trafficking, localization, and function at synapses. FITC-conjugated SEZ6L2 antibodies provide valuable tools for investigating this relationship through:

• Co-immunoprecipitation followed by fluorescence-based detection systems

• Proximity ligation assays to visualize protein-protein interactions in situ

• FRET (Fluorescence Resonance Energy Transfer) analysis when combined with differently labeled GluR1 antibodies

• Live-cell imaging to track the dynamics of SEZ6L2-GluR1 interactions

To effectively study this interaction, researchers should design experiments that:

• Compare wild-type and SEZ6L2-deficient neurons for GluR1 localization and function

• Analyze both surface and intracellular pools of AMPA receptors in relation to SEZ6L2

• Examine activity-dependent changes in the SEZ6L2-GluR1 interaction

• Assess the functional consequences of disrupting this interaction on synaptic transmission

These approaches would provide mechanistic insights into how SEZ6L2 contributes to AMPA receptor function and synaptic plasticity.

What are the optimal experimental conditions for using FITC-conjugated SEZ6L2 antibodies in flow cytometry?

When using FITC-conjugated SEZ6L2 antibodies for flow cytometry, several factors need to be optimized for reliable and reproducible results:

• Antibody titration: Perform a dilution series (typically starting from 1:100 to 1:2000) to determine the optimal concentration that provides the highest signal-to-noise ratio .

• Cell preparation: For neuronal or brain tissue samples:

  • Ensure gentle dissociation protocols to preserve membrane proteins

  • Use DNase during dissociation to prevent cell clumping

  • Include live/dead cell discrimination dyes

• Buffer composition: PBS with 1-2% BSA or FBS and 0.1% sodium azide is recommended for antibody dilution and cell resuspension .

• Staining conditions:

  • Incubate cells with antibody for 30-45 minutes at 4°C in the dark

  • Include a blocking step with 5-10% normal serum from the same species as the secondary antibody

  • Perform thorough washing steps to reduce background fluorescence

• Instrument settings:

  • FITC is optimally excited by a 488 nm laser and detected using a 530/30 nm bandpass filter

  • Perform proper compensation if using multiple fluorophores

  • Include single-color controls for accurate gating

These conditions should be further refined based on the specific cell type and experimental question being addressed.

How should researchers validate the specificity of SEZ6L2 antibodies in their experimental system?

Validating antibody specificity is crucial for ensuring reliable experimental results. For SEZ6L2 antibodies, researchers should implement a multi-faceted validation approach:

• Genetic validation:

  • Use SEZ6L2 knockout/knockdown cells or tissues as negative controls

  • Perform rescue experiments by reintroducing SEZ6L2 expression

• Molecular weight verification:

  • Confirm that the detected protein appears at the expected molecular weight (approximately 150 kDa for glycosylated SEZ6L2)

  • Consider deglycosylation treatments to verify shifts in apparent molecular weight

• Epitope blocking:

  • Pre-incubate the antibody with the immunizing peptide (if available) to demonstrate specific signal reduction

• Cross-validation:

  • Compare results using multiple antibodies targeting different epitopes of SEZ6L2

  • Correlate protein detection with mRNA expression data

• Immunoprecipitation followed by mass spectrometry:

  • Confirm the identity of the immunoprecipitated protein

These validation steps provide crucial evidence for antibody specificity and increase confidence in experimental findings.

What controls should be implemented when using SEZ6L2 antibodies in immunofluorescence studies?

For immunofluorescence studies using FITC-conjugated SEZ6L2 antibodies, the following controls are essential:

• Primary controls:

  • Positive tissue control: Brain tissue sections known to express SEZ6L2 (e.g., cerebellum, hippocampus)

  • Negative tissue control: Tissues with minimal or no SEZ6L2 expression

  • Genetic control: SEZ6L2 knockout or knockdown samples

• Technical controls:

  • Isotype control: Rabbit IgG-FITC with the same concentration as the SEZ6L2 antibody

  • Secondary antibody only control (if using indirect immunofluorescence)

  • Autofluorescence control: Unstained sample to establish background signal

• Specificity controls:

  • Peptide competition: Pre-incubation of antibody with immunizing peptide

  • Antibody titration series to determine optimal concentration

  • Cross-reactivity assessment with related proteins (SEZ6, SEZ6L)

• Imaging controls:

  • Consistent exposure settings across all samples

  • Z-stack acquisition to ensure comprehensive signal detection

  • Channel bleed-through controls when performing multi-color imaging

Implementation of these controls ensures that the observed signal is specific to SEZ6L2 and not due to technical artifacts or cross-reactivity.

How can SEZ6L2 antibodies be used to investigate the role of this protein in neurological disorders?

SEZ6L2 has been linked to various neurological and psychiatric disorders through its roles in synapse formation and function . Researchers can utilize FITC-conjugated SEZ6L2 antibodies to investigate these associations through several methodological approaches:

• Comparative expression analysis:

  • Flow cytometry to quantify SEZ6L2 expression levels in patient-derived samples vs. controls

  • Immunohistochemistry to examine altered distribution patterns in post-mortem brain tissues

• Functional studies:

  • Co-localization analysis with synaptic markers in neuronal cultures

  • Live-cell imaging to track SEZ6L2 dynamics in response to neuronal activity

• Clinical correlations:

  • Correlation of SEZ6L2 expression levels with clinical parameters or disease progression

  • Investigation of SEZ6L2 as a potential biomarker through flow cytometry of blood cells or CSF

• Therapeutic targeting assessment:

  • Monitoring changes in SEZ6L2 expression or localization in response to experimental therapies

  • Using antibodies to block SEZ6L2 function in experimental models

These applications can provide valuable insights into the pathophysiological roles of SEZ6L2 in neurological conditions and potentially identify new therapeutic targets.

What protocol modifications are necessary when using FITC-conjugated SEZ6L2 antibodies for different sample types?

Different sample types require specific modifications to protocols when using FITC-conjugated SEZ6L2 antibodies:

These modifications ensure optimal antibody performance across different experimental systems while maintaining specificity and signal intensity.

How can researchers quantitatively analyze SEZ6L2 expression using FITC-conjugated antibodies?

Quantitative analysis of SEZ6L2 expression using FITC-conjugated antibodies can be performed through several methodological approaches:

• Flow cytometry quantification:

  • Mean or median fluorescence intensity (MFI) measurements

  • Calculation of percent positive cells using appropriate gating strategies

  • Comparison with standardized beads for absolute quantification

• Fluorescence microscopy analysis:

  • Integrated density measurements of fluorescence signal

  • Cell-by-cell quantification using automated image analysis software

  • Co-localization coefficients with other markers (Pearson's or Mander's coefficients)

• High-content imaging:

  • Automated acquisition and analysis of multiple parameters

  • Machine learning approaches for complex pattern recognition

  • Correlation of SEZ6L2 expression with morphological features

• Calibration methods:

  • Use of calibration beads with known quantities of fluorophores

  • Standard curves using recombinant SEZ6L2 protein

  • Inclusion of internal standards for normalization

For accurate quantification, researchers should:

• Maintain consistent acquisition settings across all samples

• Include appropriate negative and positive controls

• Perform statistical validation of quantification methods

• Account for potential confounding factors such as autofluorescence

These approaches enable robust quantitative assessment of SEZ6L2 expression across different experimental conditions.

How can researchers address weak or absent signals when using FITC-conjugated SEZ6L2 antibodies?

When encountering weak or absent signals with FITC-conjugated SEZ6L2 antibodies, researchers should systematically troubleshoot the following aspects:

• Antibody-related factors:

  • Verify antibody viability by testing on positive control samples

  • Optimize antibody concentration through titration experiments

  • Consider photobleaching effects and minimize light exposure

  • Check antibody storage conditions and expiration dates

• Sample preparation issues:

  • Ensure adequate fixation without overfixation (which can mask epitopes)

  • Optimize antigen retrieval methods for formalin-fixed tissues

  • Verify sample integrity and protein expression

  • Adjust permeabilization conditions for intracellular epitopes

• Technical considerations:

  • Increase incubation time (e.g., overnight at 4°C instead of 1-2 hours)

  • Adjust blocking conditions to reduce background while preserving specific signals

  • Try signal amplification methods (e.g., tyramide signal amplification)

  • Consider using higher sensitivity detection systems

• Biological variables:

  • Verify SEZ6L2 expression in the specific tissue/cell type being studied

  • Consider developmental or activity-dependent regulation of expression

  • Check for potential epitope masking due to protein-protein interactions

Systematic evaluation of these factors can help identify and address the specific cause of weak or absent signals.

What strategies can be employed to differentiate between SEZ6L2 and related family members?

Distinguishing between SEZ6L2 and its related family members (SEZ6, SEZ6L) is critical for accurate experimental interpretation. Researchers can employ the following strategies:

• Epitope selection:

  • Choose antibodies targeting regions with minimal sequence homology between family members

  • Focus on the C-terminal region (AA 879-907), which has distinct sequences among SEZ6 family proteins

• Validation approaches:

  • Use knockout/knockdown models specific for each family member

  • Perform siRNA knockdown of individual family members followed by antibody testing

  • Conduct overexpression studies with tagged versions of each protein

• Analytical techniques:

  • Western blotting to distinguish based on molecular weight differences

  • Two-dimensional gel electrophoresis to separate based on both molecular weight and isoelectric point

  • Mass spectrometry identification of immunoprecipitated proteins

• Comparative analysis:

  • Co-staining with antibodies specific to each family member

  • Correlation with mRNA expression data for each family member

  • Analysis of tissue-specific expression patterns, as each family member may have distinct distribution

These approaches, used in combination, can provide robust differentiation between SEZ6L2 and related family proteins.

How can researchers optimize dual or multi-color staining protocols involving FITC-conjugated SEZ6L2 antibodies?

Optimizing multi-color staining protocols with FITC-conjugated SEZ6L2 antibodies requires careful consideration of several factors:

• Fluorophore selection:

  • Choose complementary fluorophores with minimal spectral overlap with FITC (e.g., Cy5, Alexa 647)

  • Consider the detection capabilities of available instruments

  • Account for relative brightness when pairing FITC with other fluorophores

• Staining protocol optimization:

  • Sequential staining for multiple primary antibodies from the same species

  • Appropriate blocking between steps using Fab fragments or monovalent Fab antibodies

  • Careful titration of each antibody in the multiplex panel

• Controls for multi-color experiments:

  • Single-color controls for compensation/spectral unmixing

  • Fluorescence minus one (FMO) controls to set accurate gates

  • Isotype controls for each fluorophore to assess non-specific binding

• Special considerations for brain tissue:

  • Autofluorescence quenching (using Sudan Black B or TrueBlack™)

  • Extended washing steps to reduce background

  • Appropriate antigen retrieval compatible with all target epitopes

• Image acquisition optimization:

  • Sequential scanning to minimize bleed-through

  • Adjustment of laser power and detector sensitivity for each channel

  • Consistent acquisition settings across all experimental samples

These optimization strategies enable reliable multi-parameter analysis while maintaining specificity and sensitivity for each target.

How might advances in antibody engineering impact future SEZ6L2 research?

Recent advances in antibody engineering present several opportunities for enhancing SEZ6L2 research:

• Development of recombinant antibody fragments:

  • Single-chain variable fragments (scFvs) for improved tissue penetration

  • Camelid nanobodies with unique epitope recognition properties

  • Bispecific antibodies targeting SEZ6L2 and interaction partners simultaneously

• Advanced fluorescent conjugates:

  • Quantum dot conjugation for increased photostability and brightness

  • Far-red and near-infrared fluorophores for improved tissue penetration

  • Photoactivatable or photoswitchable fluorophores for super-resolution microscopy

• Functionalized antibodies:

  • Cell-penetrating antibodies for live-cell imaging of intracellular domains

  • Conformation-specific antibodies to distinguish active versus inactive SEZ6L2

  • Proximity-labeling antibodies to identify novel interaction partners

• Therapeutic applications:

  • Development of humanized antibodies for potential clinical applications

  • Antibody-drug conjugates targeting SEZ6L2 in overexpressing tumors

  • Intrabodies for specific subcellular targeting

These advances would expand the toolkit available for SEZ6L2 research and potentially lead to new therapeutic strategies targeting this protein.

What emerging methodologies could enhance our understanding of SEZ6L2 function and regulation?

Several emerging methodologies hold promise for advancing our understanding of SEZ6L2:

• Advanced imaging techniques:

  • Live STED or STORM super-resolution microscopy to visualize SEZ6L2 nanoscale organization

  • Lattice light-sheet microscopy for long-term, non-phototoxic imaging of SEZ6L2 dynamics

  • Expansion microscopy to physically enlarge specimens for enhanced resolution

• Single-cell approaches:

  • Single-cell transcriptomics combined with protein detection (CITE-seq)

  • Mass cytometry (CyTOF) with metal-conjugated SEZ6L2 antibodies

  • Spatial transcriptomics to correlate SEZ6L2 protein with local gene expression

• Protein interaction studies:

  • BioID or APEX2 proximity labeling to identify the SEZ6L2 interactome

  • FRET-FLIM for quantitative analysis of protein-protein interactions in situ

  • Split fluorescent protein complementation for visualizing interactions in living cells

• Functional genomics:

  • CRISPR-Cas9 screening to identify genes affecting SEZ6L2 function

  • CRISPR activation/inhibition to modulate SEZ6L2 expression

  • Base editing for introducing specific mutations in SEZ6L2

These methodologies would provide unprecedented insights into SEZ6L2 molecular functions, regulation, and involvement in both physiological and pathological processes.

How should researchers interpret discrepancies in SEZ6L2 molecular weight across different experimental conditions?

Variations in observed SEZ6L2 molecular weight are common across different experimental conditions and can provide valuable biological insights if properly interpreted:

• Glycosylation effects:

  • The calculated molecular weight of SEZ6L2 is approximately 98 kDa, but glycosylation typically increases the observed weight to around 150 kDa

  • Tissue-specific glycosylation patterns may result in different apparent molecular weights

  • Treatment with glycosidases (PNGase F, Endo H) can confirm glycosylation as the source of weight variation

• Protein processing considerations:

  • Proteolytic cleavage may generate fragments with lower molecular weights

  • Alternative splicing could produce isoforms with different sizes

  • Post-translational modifications beyond glycosylation (phosphorylation, ubiquitination) can alter migration patterns

• Technical variables affecting migration:

  • Gel percentage and electrophoresis conditions can impact apparent molecular weight

  • Reducing vs. non-reducing conditions may reveal different conformations

  • Sample preparation methods (heating, detergent types) can affect protein denaturation

• Analytical framework for interpretation:

  • Compare with recombinant protein standards when available

  • Cross-reference with mass spectrometry data when possible

  • Consider species-specific variations in post-translational modifications

Understanding these factors enables researchers to correctly interpret molecular weight variations and extract valuable information about SEZ6L2 processing and modification in different biological contexts.

What statistical approaches are most appropriate for analyzing SEZ6L2 expression data from flow cytometry experiments?

When analyzing SEZ6L2 expression data from flow cytometry experiments using FITC-conjugated antibodies, researchers should consider the following statistical approaches:

• Descriptive statistics:

  • Mean/median fluorescence intensity (MFI) with standard deviation/interquartile range

  • Coefficient of variation to assess population homogeneity

  • Percent positive cells above threshold determined by appropriate controls

• Comparative analyses:

  • Student's t-test or Mann-Whitney U test for two-group comparisons

  • ANOVA or Kruskal-Wallis for multiple group comparisons

  • Repeated measures ANOVA for longitudinal studies

  • Appropriate post-hoc tests with correction for multiple comparisons

• Advanced analytical approaches:

  • Multivariate analysis for complex phenotyping (PCA, t-SNE, UMAP)

  • Hierarchical clustering to identify sample relationships

  • Machine learning algorithms for pattern recognition

  • Correlation analyses between SEZ6L2 expression and other parameters

• Specific considerations for SEZ6L2 analysis:

  • Account for autofluorescence, particularly in brain-derived samples

  • Consider normalization to housekeeping proteins for relative quantification

  • Implement appropriate transformation for non-normally distributed data

  • Establish clear criteria for defining SEZ6L2-positive populations

These statistical approaches, when properly applied, enable robust analysis of SEZ6L2 expression patterns and their correlation with biological or clinical parameters.