SEMA6B Antibody, HRP conjugated

What is SEMA6B and what specific applications is SEMA6B Antibody, HRP conjugated optimized for?

SEMA6B (Semaphorin 6B) is a single-pass type-I transmembrane protein belonging to the semaphorin axon-guidance family. It contains a sema domain, transmembrane domain (TM), and cytoplasmic domain . SEMA6B plays crucial roles in neural development by functioning primarily as a repellent for sympathetic ganglion axons and inhibiting neurite outgrowth through interaction with receptor plexin-A4 .

SEMA6B Antibody, HRP conjugated has been specifically optimized for ELISA applications. According to product specifications, the HRP-conjugated variant (CSB-PA862010LB01HU) is primarily recommended for ELISA techniques, while non-conjugated versions of the antibody may be suitable for additional applications including immunohistochemistry (IHC) and immunofluorescence (IF) .

What are the recommended dilution ratios for SEMA6B antibodies in different experimental applications?

The optimal dilution ratios vary by application technique:

For HRP-conjugated antibodies specifically used in ELISA, a standardized protocol is provided in the GENLISA Human Semaphorin 6B ELISA kit, which utilizes a sandwich ELISA technique with double antibodies to ensure high sensitivity and specificity .

What expression patterns of SEMA6B should researchers expect in different tissue types?

SEMA6B is predominantly expressed in:

• Adult brain - particularly in neurons where staining is localized to both membrane and cytoplasm

• Heart tissue

• Liver tissues - recent studies have identified SEMA6B expression in human liver tissues, especially in inflammatory conditions like HBV-related acute-on-chronic liver failure

• Colorectal tissues - with significantly higher expression in colorectal cancer tissues compared to normal colorectal tissues

In cellular distribution studies, wild-type SEMA6B typically localizes to the plasma membrane, whereas truncated forms (such as those produced by disease-causing mutations) may show more homogeneous distribution throughout the cell and fail to properly localize to the membrane .

How should researchers validate the specificity of SEMA6B Antibody, HRP conjugated in their experimental systems?

A comprehensive validation approach should include:

• Positive controls: Use tissues with known high SEMA6B expression (brain cortex, colorectal cancer samples) as positive controls

• Negative controls:

  • Omission of primary antibody

  • Use of tissues known to have low/no SEMA6B expression

  • SEMA6B knockdown samples via RNA interference

• Western blot analysis: Confirm antibody specificity by detecting a band at the expected molecular weight (95 kDa)

• Cross-reactivity assessment: Test reactivity against other semaphorin family members, particularly those with high sequence homology to SEMA6B

• Recombinant protein competition: Pre-incubation with recombinant SEMA6B protein should abolish specific staining

Recent studies have demonstrated that SEMA6B-specific antibodies can detect both the full-length protein (95.3 kDa) and truncated variants resulting from disease-associated mutations .

What are the optimal storage conditions and handling procedures for maintaining SEMA6B Antibody, HRP conjugated activity?

To maintain optimal activity of SEMA6B Antibody, HRP conjugated:

• Storage temperature: Store at -20°C for long-term storage

• Buffer composition: The antibody is typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Stability: Antibodies stored properly are typically stable for one year after shipment

• Aliquoting: For -20°C storage, aliquoting may be unnecessary, but for frequent use, dividing into small aliquots is recommended to avoid repeated freeze-thaw cycles

• Working dilutions: Prepare fresh working dilutions on the day of use

• HRP consideration: As HRP is sensitive to bacterial contamination and some metal ions, ensure sterile handling and avoid metal containers

These conditions are critical for maintaining both antibody specificity and HRP enzymatic activity, which directly impacts assay sensitivity and reproducibility .

How can SEMA6B Antibody, HRP conjugated be utilized to study pathogenic SEMA6B variants associated with neurological disorders?

Recent research has identified several pathogenic variants in SEMA6B associated with progressive myoclonic epilepsy (PME) and intellectual disability (ID) . SEMA6B Antibody, HRP conjugated can be employed to study these variants through:

• Expression analysis: Quantitative ELISA to determine if pathogenic variants alter SEMA6B expression levels in patient samples or model systems

• Functional studies in cellular models:

  • Generate cell lines expressing wild-type or mutant SEMA6B

  • Use HRP-conjugated antibodies in ELISA to quantify differential protein expression

  • Compare results with western blotting using non-conjugated antibodies

• Protein interaction studies:

  • Develop sandwich ELISA systems using captured recombinant SEMA6B interaction partners (like Plexin A4)

  • Use HRP-conjugated SEMA6B antibodies to detect binding differences between wild-type and mutant variants

Studies have shown that truncating variants (p.Gln686*) result in proteins with remarkably increased expression compared to wild-type, while missense variants (p.Gly495Trp) may maintain normal expression levels but exhibit altered tertiary structures and protein-binding properties .

What methodological considerations are important when using SEMA6B Antibody, HRP conjugated in multiplex immunoassays?

When incorporating SEMA6B Antibody, HRP conjugated into multiplex immunoassays:

• Cross-reactivity assessment:

  • Test for potential cross-reactivity with other antibodies in the multiplex panel

  • Evaluate whether HRP signal interferes with other detection systems (e.g., fluorescence)

• Signal optimization:

  • Determine optimal antibody concentration to prevent signal saturation

  • Establish standard curves using recombinant SEMA6B protein to ensure quantitative accuracy

• Blocking optimization:

  • Use appropriate blocking agents to minimize non-specific binding

  • Consider QuickBlock for efficient blocking as suggested in recent protocols

• Substrate selection:

  • For multiplex colorimetric detection, choose substrate combinations with minimal spectral overlap

  • When using TMB substrate with HRP-conjugated antibodies, ensure the reaction is adequately stopped before measurement at 450nm

• Validation controls:

  • Include tissue-specific positive and negative controls

  • Incorporate single-antibody controls to identify any cross-reactivity issues

These considerations help ensure accurate, specific detection in complex multiplex systems where multiple biological targets are simultaneously assessed .

How can researchers effectively use SEMA6B Antibody, HRP conjugated to investigate SEMA6B's role in tumor progression and cancer?

SEMA6B has been implicated in tumor progression, particularly in colorectal cancer where its overexpression correlates with poor prognosis . To investigate this role using SEMA6B Antibody, HRP conjugated:

• Expression profiling in cancer tissues:

  • Develop quantitative ELISA assays using HRP-conjugated antibodies to measure SEMA6B levels in tumor versus normal tissues

  • Correlate expression levels with clinical outcomes and pathological features

• Immune microenvironment analysis:

  • Use ELISA to quantify SEMA6B in relation to immune cell infiltration markers

  • Recent studies showed SEMA6B expression positively correlates with stromal scores, immune scores, and ESTIMATE scores in colorectal cancer

• Functional studies:

  • Implement knockdown/overexpression studies and measure resulting SEMA6B levels using ELISA

  • In colorectal cancer, SEMA6B knockdown has been shown to significantly reduce cell proliferation and migration

• Pathway analysis:

  • Combine with other antibodies to investigate SEMA6B's involvement in signaling pathways

  • Gene Set Enrichment Analysis (GSEA) has revealed SEMA6B's association with immune-related processes and cancer signaling pathways

• Therapeutic response monitoring:

  • Develop ELISA assays to monitor SEMA6B levels before and after treatment

  • Evaluate SEMA6B as a potential biomarker for treatment response

This approach has revealed that SEMA6B expression correlates with infiltration levels of macrophages, MDSCs, NK cells, Tregs, and Th1 cells in colorectal cancer, suggesting its potential as a therapeutic target .

What are common technical challenges when using SEMA6B Antibody, HRP conjugated, and how can they be addressed?

Addressing these challenges requires systematic optimization of each experimental parameter, beginning with the manufacturer's recommended protocols and adjusting based on specific experimental conditions .

How can researchers effectively compare results obtained using SEMA6B Antibody, HRP conjugated with those from other detection methods?

When comparing results across different detection methods:

• Standardization:

  • Use recombinant SEMA6B proteins as standards across all methods

  • Develop standard curves for quantitative comparisons

  • Include identical positive and negative controls across platforms

• Cross-validation approaches:

  • Compare ELISA results (using HRP-conjugated antibodies) with western blot data (using non-conjugated antibodies)

  • Validate findings with orthogonal methods such as RT-qPCR for mRNA expression

  • Consider immunofluorescence or immunohistochemistry for spatial context

• Correlation analysis:

  • Perform statistical correlation analyses between different methods

  • Establish conversion factors if systematic differences exist

  • Document method-specific sensitivity and detection limits

• Discrepancy resolution:

  • When results differ between methods, consider:

    • Epitope accessibility differences

    • Post-translational modifications

    • Protein conformation variations

    • Method-specific technical limitations

• Data normalization strategies:

  • Use housekeeping proteins (β-actin) as internal controls across methods

  • Apply the 2−ΔΔCt method for comparing relative expression levels

  • Consider using digital pathology tools for standardized quantification of immunohistochemistry results

How can SEMA6B Antibody, HRP conjugated be employed to study SEMA6B's role in neurological disorders?

SEMA6B has been recently implicated in progressive myoclonic epilepsy (PME) and intellectual disability (ID) . Researchers can use SEMA6B Antibody, HRP conjugated to investigate these conditions through:

• Patient sample analysis:

  • Develop quantitative ELISA assays to measure SEMA6B levels in cerebrospinal fluid or blood samples from affected individuals

  • Compare protein levels between patients with different SEMA6B variants and control subjects

• Neuronal culture studies:

  • Quantify SEMA6B expression in primary neuronal cultures under various conditions

  • Assess the impact of disease-associated variants on protein expression and localization

• Disease mechanism investigation:

  • Implement sandwich ELISA to study interactions between SEMA6B and its binding partners (e.g., PlxnA2)

  • Recent co-immunoprecipitation studies have shown that both truncating and missense variants influence protein binding of SEMA6B and PlxnA2 with varying degrees

• Therapeutic development:

  • Screen potential therapeutic compounds that might restore normal SEMA6B function

  • Monitor changes in SEMA6B expression during treatment trials

• Biomarker development:

  • Evaluate SEMA6B as a potential biomarker for disease progression or treatment response

  • Correlate SEMA6B levels with clinical parameters and neuroimaging findings

Recent research has identified that variants in SEMA6B can lead to subcellular mislocalization, reduced spine density, and impaired axon guidance, providing multiple potential avenues for therapeutic intervention .

What protocols have been developed for using SEMA6B Antibody, HRP conjugated in studying liver diseases?

Recent research has identified SEMA6B's role in hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) . The following protocols have been developed:

• Quantitative ELISA for patient samples:

  • Extraction of peripheral blood mononuclear cells (PBMCs) using density gradient centrifugation

  • ELISA quantification using SEMA6B Antibody, HRP conjugated

  • Comparison between patient groups (HBV-ACLF, liver cirrhosis, chronic hepatitis B, and normal controls)

• Liver tissue expression analysis:

  • Preparation of liver tissue sections (4 μm) from paraffin-embedded samples

  • Antigen retrieval using microwave with Tris-EDTA buffer (pH 9) or Citrate buffer (pH 6)

  • Blocking with 5% bovine serum albumin (BSA)

  • Incubation with primary antibody against SEMA6B, followed by HRP-conjugated secondary antibody

  • Visualization using an HRP kit and counterstaining with hematoxylin

  • Quantification of SEMA6B-positive cells using Image-Pro Plus software

• Cell culture models:

  • SEMA6B expression analysis in hepatocyte cell lines (HepaRG) and macrophages (THP1)

  • Creation of SEMA6B knockdown and wild-type models

  • Stimulation with lipopolysaccharide (LPS) to mimic inflammatory conditions

  • Western blot and qRT-PCR validation of SEMA6B expression levels

  • Functional assays including cell cycle analysis, proliferation assays, and apoptosis assessment

These protocols have revealed that SEMA6B induces macrophage-mediated inflammation and hepatocyte apoptosis in HBV-ACLF, suggesting its potential as a therapeutic target .

What emerging applications for SEMA6B Antibody, HRP conjugated should researchers consider exploring?

Emerging research suggests several promising future applications:

• Single-cell analysis:

  • Development of ultrasensitive ELISA protocols for detecting SEMA6B in single cells

  • Integration with single-cell RNA-seq data to correlate protein and mRNA levels at single-cell resolution

  • Recent studies have already begun exploring cell types expressing SEMA6B in single-cell RNA-seq data from healthy liver tissues

• Liquid biopsy development:

  • Optimization of ELISA protocols for detecting circulating SEMA6B in blood or other body fluids

  • Evaluation as a non-invasive biomarker for neurological disorders or cancer progression

  • Correlation with disease severity and treatment response

• Therapeutic antibody development:

  • Using structural insights from HRP-conjugated antibody binding studies to develop therapeutic antibodies targeting SEMA6B

  • Creation of antibody-drug conjugates for targeted delivery to SEMA6B-expressing cells

  • Development of bispecific antibodies targeting SEMA6B and its receptors

• Neurodevelopmental research:

  • Quantitative assessment of SEMA6B expression during critical periods of brain development

  • Correlation with structural and functional brain development parameters

  • Investigation of SEMA6B's role in synaptogenesis and neural circuit formation

• Cancer immunotherapy applications:

  • Exploration of SEMA6B as an immunotherapy target based on its correlation with immune cell infiltration

  • Development of protocols to monitor SEMA6B levels during immunotherapy

  • Recent research has shown positive correlations between SEMA6B expression and infiltration levels of various immune cells in colorectal cancer

These emerging applications highlight SEMA6B's potential significance in both diagnostic and therapeutic contexts across multiple disease areas .

How might advances in antibody engineering impact future iterations of SEMA6B Antibody, HRP conjugated?

Future advances in antibody engineering will likely enhance SEMA6B Antibody, HRP conjugated in several ways:

• Increased specificity and sensitivity:

  • Development of recombinant antibodies with higher affinity and specificity

  • Application of phage display or yeast display technologies to select optimal binding domains

  • Generation of antibodies against specific SEMA6B epitopes relevant to disease states

• Enhanced conjugation chemistry:

  • Site-specific conjugation of HRP to maintain optimal antibody orientation

  • Development of novel linkers with improved stability and reduced steric hindrance

  • Controllable enzyme-to-antibody ratios for optimized signal generation

• Multimodal detection capabilities:

  • Creation of dual-labeled antibodies combining HRP with fluorescent markers for orthogonal detection

  • Development of antibodies compatible with both solution-based and tissue-based assays

  • Integration with emerging biosensor technologies

• Improved stability and shelf-life:

  • Engineering antibodies with enhanced thermostability

  • Development of lyophilized formulations with extended shelf-life

  • Reduction or elimination of sodium azide in storage buffers

• Humanized research antibodies:

  • Development of humanized versions for potential therapeutic applications

  • Reduced immunogenicity for in vivo applications

  • Closer structural similarity to naturally occurring antibodies

These advances will likely enable more precise and versatile applications of SEMA6B antibodies in both research and clinical settings, facilitating better understanding of SEMA6B's roles in health and disease .