TRABD2B Antibody, HRP conjugated

What is TRABD2B and what function does it serve in cellular pathways?

TRABD2B (also known as Metalloprotease TIKI2) is a 517 amino acid metalloproteinase that functions as a negative regulator of Wnt signaling pathways. It mediates the cleavage of N-terminal residues in many Wnt proteins, thereby modulating their activity. Unlike its counterpart TIKI1 (which is required for head formation via Wnt cleavage-oxidation and inactivation), TIKI2 operates through specific metalloprotease activity that is dependent on Mn²⁺/Co²⁺ and can be inhibited by divalent metal chelators such as EDTA. In pathological contexts, TRABD2B has been found to be upregulated in renal cell carcinoma while appearing to suppress growth in osteosarcoma by targeting the canonical Wnt pathway .

What are the key specifications of commercially available TRABD2B Antibody, HRP conjugated?

The commercially available TRABD2B Antibody, HRP conjugated is a polyclonal antibody raised in rabbits using recombinant Human Metalloprotease TIKI2 protein (amino acids 195-405) as the immunogen. It is specifically reactive against human TRABD2B and has been validated for ELISA applications. The antibody is provided in liquid form with a storage buffer containing 0.03% Proclin 300 preservative, 50% glycerol, and 0.01M PBS at pH 7.4. It has been purified using antigen affinity chromatography and is of IgG isotype .

How should TRABD2B Antibody, HRP conjugated be stored to maintain optimal activity?

For optimal preservation of antibody activity, TRABD2B Antibody, HRP conjugated should be stored at -20°C or -80°C upon receipt. It is critical to avoid repeated freeze-thaw cycles as these can significantly compromise antibody function and sensitivity. For longer-term storage planning, the antibody has been demonstrated to maintain stability for up to 12 months from the date of receipt when stored at -20°C to -70°C. For projects requiring frequent access to the antibody, aliquoting is strongly recommended to minimize freeze-thaw cycles. When stored under sterile conditions after reconstitution, the antibody maintains stability for approximately 1 month at 2-8°C or 6 months at -20°C to -70°C .

What experimental controls should be included when using TRABD2B Antibody, HRP conjugated in Western blot applications?

When designing Western blot experiments using TRABD2B Antibody, HRP conjugated, several controls are essential for result validation:

• Positive Control: Use lysates from HEK293 cells transfected with human TIKI2/TRABD2B. This provides confirmation of antibody specificity and expected band size (approximately 65 kDa under reducing conditions) .

• Negative Control: Include mock-transfected HEK293 cells to demonstrate specificity and absence of non-specific binding .

• Loading Control: Incorporate antibodies against housekeeping proteins such as GAPDH or β-actin to normalize protein loading.

• Secondary Antibody Control: Run a lane with sample but without primary antibody to detect any non-specific binding of the secondary antibody.

• Molecular Weight Marker: Include a protein ladder to accurately identify the TRABD2B band at approximately 65 kDa.

For optimal signal detection, a concentration of 0.5 μg/mL of the antibody is recommended, though this should be optimized for your specific experimental conditions .

How can TRABD2B Antibody, HRP conjugated be effectively used in ELISA applications?

For optimal utilization of TRABD2B Antibody, HRP conjugated in ELISA applications, follow these methodological considerations:

• Coating Optimization: For direct ELISA, coat plates with purified TRABD2B protein (ideally using the recombinant human Metalloprotease TIKI2 protein spanning amino acids 195-405) at concentrations ranging from 0.1-1.0 μg/well.

• Antibody Dilution Range: Begin with dilutions between 1:1000 and 1:5000 for the HRP-conjugated antibody. The optimal concentration should be determined empirically through a dilution series for each specific experimental setup .

• Blocking Protocol: Use 2-5% BSA or non-fat milk in PBS to reduce background signal. Incubate for 1-2 hours at room temperature.

• Detection System: Since the antibody is already HRP-conjugated, use a suitable substrate such as TMB (3,3',5,5'-Tetramethylbenzidine) for colorimetric detection or luminol-based reagents for enhanced chemiluminescence.

• Standard Curve Preparation: Include a standard curve using recombinant TRABD2B protein at concentrations ranging from 0-1000 ng/mL to quantify unknown samples.

Remember that optimal dilutions and protocols should be determined by each laboratory for their specific application and experimental conditions .

What methodological approaches can be used to investigate TRABD2B's role in the Wnt signaling pathway?

To investigate TRABD2B's role in Wnt signaling, researchers can employ several complementary approaches:

• Enzymatic Activity Assays: Assess TRABD2B's metalloprotease activity by incubating purified TRABD2B with Wnt proteins and analyzing cleavage products via mass spectrometry or Western blotting. Include controls with metalloprotease inhibitors like EDTA to confirm specificity .

• TOPFlash Reporter Assays: Measure β-catenin-dependent transcriptional activity using TOPFlash luciferase reporter constructs in cells with modulated TRABD2B expression. This quantifies the functional impact of TRABD2B on canonical Wnt signaling output .

• Co-immunoprecipitation Studies: Use anti-TRABD2B antibodies to pull down protein complexes and identify Wnt pathway components that physically interact with TRABD2B.

• Cell-based Phenotypic Assays: In cell types where Wnt signaling drives specific phenotypes (e.g., osteoblast differentiation), assess how TRABD2B overexpression or knockdown affects these outcomes.

• CRISPR-Cas9 Gene Editing: Generate TRABD2B knockout cell lines to comprehensively assess pathway changes in the absence of TRABD2B function.

These approaches can be combined with the use of HRP-conjugated TRABD2B antibodies for protein detection and quantification throughout experimental workflows.

How can TRABD2B Antibody, HRP conjugated be utilized in flow cytometry for detecting TRABD2B expression in transfected cell lines?

While the HRP-conjugated version of the TRABD2B antibody is not optimal for flow cytometry due to the nature of the HRP conjugate, researchers can adapt protocols based on unconjugated TRABD2B antibody methodologies. Based on the available data with other TRABD2B antibody formats:

• Cell Preparation: Harvest HEK293 cells transfected with human TIKI2/TRABD2B and untransfected controls. Wash cells in flow cytometry buffer (PBS with 2% FBS).

• Antibody Adaptation: For the HRP-conjugated antibody, a modified protocol is required: first fix and permeabilize cells to allow intracellular staining if the target epitope is not surface-exposed.

• Signal Development: After antibody incubation, cells must be washed thoroughly and incubated with a fluorogenic HRP substrate compatible with flow cytometry (such as PerCP, which produces fluorescence upon HRP activity).

• Gating Strategy: Use forward and side scatter to identify the cell population of interest, and then analyze fluorescence intensity to quantify TRABD2B expression.

• Controls: Include mock-transfected cells as negative controls to establish baseline fluorescence and determine positive staining thresholds .

For more standardized flow cytometry protocols, researchers might consider using specifically designed fluorochrome-conjugated antibodies rather than HRP-conjugated versions.

What are the technical considerations for using TRABD2B Antibody, HRP conjugated in investigating the relationship between TRABD2B expression and cancer progression?

When investigating the relationship between TRABD2B expression and cancer progression using TRABD2B Antibody, HRP conjugated, researchers should consider several technical aspects:

• Tissue Sample Preparation: For cancer tissue microarrays or patient-derived samples, optimize fixation conditions (typically 10% neutral buffered formalin) and antigen retrieval methods to ensure TRABD2B epitope accessibility.

• Expression Quantification: Develop a standardized scoring system for immunohistochemistry to quantify TRABD2B expression levels across different cancer stages and grades. This can involve H-score methods (intensity × percentage of positive cells) or automated image analysis software.

• Correlation Analysis: Design experiments to correlate TRABD2B expression levels with:

  • Clinical parameters (tumor stage, grade, patient survival)

  • Molecular markers of Wnt pathway activation

  • Known prognostic indicators specific to the cancer type (especially for renal cell carcinoma and osteosarcoma where TRABD2B has known roles)

• Functional Validation: Complement expression studies with functional assays in relevant cancer cell lines, manipulating TRABD2B levels and measuring effects on proliferation, migration, and invasion.

• Technical Controls: For each experiment, include positive controls (tissues known to express TRABD2B) and negative controls (antibody diluent only) to validate staining specificity.

How can researchers optimize dual immunofluorescence protocols using TRABD2B Antibody, HRP conjugated alongside other markers?

Optimizing dual immunofluorescence protocols using TRABD2B Antibody, HRP conjugated with other molecular markers requires careful consideration of several technical factors:

• HRP Conversion System: Since the antibody is HRP-conjugated, researchers need to employ a tyramide signal amplification (TSA) system to convert HRP activity into fluorescence:

  • Use tyramide-conjugated fluorophores (e.g., tyramide-FITC or tyramide-Cy3)

  • The HRP catalyzes covalent binding of the fluorophore to proteins in the vicinity of the antibody

• Sequential Staining Protocol:

  • Perform TRABD2B staining first using the HRP-conjugated antibody

  • Develop with appropriate tyramide-fluorophore

  • Thoroughly quench the HRP activity using 3% hydrogen peroxide

  • Proceed with the second primary antibody (from a different host species)

  • Detect using a directly conjugated fluorescent secondary antibody

• Epitope Considerations:

  • When combining with Wnt pathway markers, prioritize antibodies targeting different regions of the signaling cascade

  • Consider detergent concentration and antigen retrieval methods that will work for both antigens

• Spectral Compatibility:

  • Select fluorophores with minimal spectral overlap to avoid bleed-through

  • Include single-stained controls to confirm specificity and absence of cross-reactivity

• Image Acquisition Parameters:

  • Use sequential scanning rather than simultaneous detection when using confocal microscopy

  • Establish exposure settings using control samples to prevent signal saturation

What are the most common causes of high background when using TRABD2B Antibody, HRP conjugated, and how can they be addressed?

When encountering high background with TRABD2B Antibody, HRP conjugated, several common causes and solutions should be considered:

For particularly persistent background issues, consider implementing a stringent wash protocol using PBS-T (0.1% Tween-20) followed by high-salt washes (PBS with 0.5M NaCl) to disrupt non-specific ionic interactions .

How should researchers interpret discrepancies between ELISA and Western blot results when using TRABD2B Antibody, HRP conjugated?

When facing discrepancies between ELISA and Western blot results using TRABD2B Antibody, HRP conjugated, researchers should consider several factors in their analysis:

• Epitope Accessibility Differences:

  • ELISA typically uses native or partially denatured proteins where certain epitopes may be masked

  • Western blot uses fully denatured proteins where linear epitopes are exposed

  • If the antibody targets amino acids 195-405 of TRABD2B, structural changes could affect recognition

• Post-translational Modifications:

  • Western blot may reveal multiple bands due to different glycosylation states or proteolytic processing

  • ELISA might preferentially detect certain modified forms based on epitope availability

• Sample Preparation Considerations:

  • Cell lysis buffers for Western blot may preserve or disrupt certain protein interactions

  • Coating buffers for ELISA may partially denature proteins affecting antibody binding

• Quantitative Analysis Approach:

  • Create a standardized calibration curve using recombinant TRABD2B protein

  • Normalize Western blot data to housekeeping proteins

  • Compare relative rather than absolute values between techniques

• Validation Strategy:

  • Use alternative antibodies targeting different TRABD2B epitopes

  • Perform knockdown/overexpression experiments to confirm specificity

  • Consider mass spectrometry to identify proteins detected in conflicting results

When interpreting divergent results, consider that both methods provide complementary information: Western blot reveals size and potential modifications while ELISA provides more precise quantification under native conditions.

What experimental strategies can address potential cross-reactivity of TRABD2B Antibody, HRP conjugated with other TRAB domain-containing proteins?

To address potential cross-reactivity of TRABD2B Antibody with other TRAB domain-containing proteins, researchers should implement the following experimental strategies:

• Competitive Inhibition Assays:

  • Pre-incubate the antibody with excess recombinant TRABD2B protein (195-405 AA)

  • Compare staining patterns with and without competitive inhibition

  • Specific binding should be significantly reduced or eliminated in the presence of excess antigen

• Genetic Validation Approaches:

  • Test antibody reactivity in TRABD2B knockout cell lines generated via CRISPR-Cas9

  • Compare with TRABD2B-overexpressing cells as positive controls

  • Any persistent signal in knockout cells suggests cross-reactivity

• Bioinformatic Analysis and Testing:

  • Identify sequence homology between TRABD2B and other TRAB domain proteins (particularly TRABD and TRABD2A)

  • Test antibody against recombinant proteins of these family members

  • Create a cross-reactivity profile detailing relative affinity for each protein

• Immunoprecipitation-Mass Spectrometry:

  • Use the antibody for immunoprecipitation followed by mass spectrometry

  • Identify all proteins pulled down by the antibody

  • Quantify the relative abundance of TRABD2B versus other detected proteins

• Epitope Mapping:

  • Generate a series of peptide fragments covering the immunogen region

  • Determine which specific sequences within the 195-405 AA region are recognized

  • Compare these epitopes with sequence alignments of related proteins

By systematically implementing these approaches, researchers can definitively characterize antibody specificity and account for any cross-reactivity in experimental interpretations.

How does TRABD2B Antibody, HRP conjugated compare with other detection systems for studying TIKI2's role in Wnt signaling modulation?

When comparing TRABD2B Antibody, HRP conjugated with alternative detection systems for studying TIKI2's role in Wnt signaling modulation, several factors should be considered:

For comprehensive Wnt signaling studies, researchers should consider combining these approaches. For instance, use HRP-conjugated antibodies for quantitative analysis in ELISA and Western blot, while employing fluorescent approaches for subcellular localization studies. The specific experimental question should guide selection of the most appropriate detection system .

What are the methodological differences between using sheep anti-human TRABD2B antibodies versus rabbit polyclonal antibodies for TRABD2B detection?

The methodological differences between sheep anti-human TRABD2B antibodies and rabbit polyclonal antibodies for TRABD2B detection involve several important technical considerations:

• Host Species Considerations:

  • Sheep antibodies offer advantages in multiplex staining with rabbit and mouse primary antibodies

  • Rabbit polyclonals typically have wider secondary antibody availability and compatibility with common detection systems

• Epitope Recognition Patterns:

  • Sheep anti-human TRABD2B (e.g., R&D Systems AF1600) demonstrated specific detection at ~65 kDa in Western blot

  • Rabbit polyclonal antibodies (like those from Cusabio) use immunogen from amino acids 195-405, which may recognize different epitopes within TRABD2B

• Application Optimization Requirements:

  • Sheep antibodies typically require higher concentrations (0.5 μg/mL recommended for Western blot)

  • Rabbit polyclonals often work at higher dilutions (1:1000-1:2000) for comparable applications

• Signal Development Systems:

  • For the HRP-conjugated rabbit polyclonal, direct substrate addition is sufficient

  • Sheep antibodies require an additional secondary antibody incubation step with anti-sheep IgG-HRP

• Cross-Reactivity Profiles:

  • Sheep antibodies may have different non-specific binding profiles than rabbit antibodies

  • This can be advantageous when working in tissues with high endogenous rabbit IgG

When selecting between these options, researchers should consider the specific experimental design, available detection systems, and potential for cross-reactivity with other proteins in their experimental system .

What alternative approaches can researchers use when TRABD2B Antibody, HRP conjugated demonstrates limited sensitivity in detecting endogenous TRABD2B in specific cell types?

When encountering limited sensitivity with TRABD2B Antibody, HRP conjugated for detecting endogenous TRABD2B in specific cell types, researchers can implement several alternative approaches:

• Signal Amplification Technologies:

  • Employ tyramide signal amplification (TSA) to enhance HRP-based detection

  • This catalytically deposits multiple tyramide-conjugated fluorophores or chromogens at the antibody binding site

  • Can increase sensitivity by 10-100 fold compared to conventional detection

• Alternative Detection Platforms:

  • Switch to highly sensitive chemiluminescent substrates (SuperSignal West Femto)

  • Consider digital immunoassay platforms like Simoa or Single Molecule Counting that can detect proteins at femtomolar concentrations

  • Explore proximity ligation assay (PLA) which can detect single protein molecules through rolling circle amplification

• Sample Enrichment Strategies:

  • Perform immunoprecipitation to concentrate TRABD2B before analysis

  • Use subcellular fractionation to isolate membrane fractions where TRABD2B is likely concentrated

  • Implement cell sorting to isolate subpopulations with higher TRABD2B expression

• Transcriptional Analysis Complementation:

  • Combine protein detection with RT-qPCR to assess TRABD2B mRNA levels

  • Use RNA-scope in situ hybridization for sensitive detection of TRABD2B transcripts in tissue sections

  • Correlate transcript and protein data to validate low expression findings

• Genetic Tagging Approaches:

  • For cultured cells, consider CRISPR knock-in of epitope tags (FLAG, HA, etc.) to the endogenous TRABD2B locus

  • Use well-characterized tag-specific antibodies with established high sensitivity

  • This maintains endogenous expression levels while improving detection sensitivity

By implementing these alternative strategies, researchers can overcome sensitivity limitations while still addressing their scientific questions regarding TRABD2B expression and function in challenging experimental systems .

How can researchers design experiments using TRABD2B Antibody, HRP conjugated to investigate the metalloprotease activity of TIKI2 on specific Wnt proteins?

To investigate TIKI2's metalloprotease activity on specific Wnt proteins using TRABD2B Antibody, HRP conjugated, researchers can implement the following experimental design:

• In Vitro Cleavage Assay Development:

  • Express and purify recombinant Wnt proteins with N-terminal tags

  • Incubate with immunoprecipitated TRABD2B (using the antibody)

  • Analyze cleavage products via Western blot or mass spectrometry

  • Include controls with metalloprotease inhibitors (EDTA) and catalytically inactive TRABD2B mutants

• Substrate Specificity Profiling:

  • Test multiple Wnt family members in parallel cleavage reactions

  • Quantify relative cleavage efficiency using densitometry of Western blots

  • Correlate cleavage patterns with Wnt protein sequence alignment to identify recognition motifs

  • Design synthetic peptide substrates based on identified cleavage sites for kinetic studies

• Cell-Based Validation Approach:

  • Co-express TRABD2B and various Wnt proteins in HEK293 cells

  • Immunoprecipitate Wnt proteins from conditioned media and cell lysates

  • Analyze processing status using antibodies specific to N-terminal and C-terminal regions

  • Correlate processing with Wnt signaling activity using TOPFlash reporter assays

• Kinetic Parameter Determination:

  • Design FRET-based peptide substrates containing identified TRABD2B cleavage sites

  • Measure reaction kinetics with purified TRABD2B enzyme

  • Determine Km and kcat values for different Wnt-derived substrates

  • Analyze the effect of divalent cations (Mn²⁺/Co²⁺) on enzyme activity

This comprehensive approach will provide mechanistic insights into how TRABD2B/TIKI2 regulates Wnt signaling through its metalloprotease activity .

What methodological considerations are important when studying the role of TRABD2B in cancer progression using tissue microarrays?

When studying TRABD2B's role in cancer progression using tissue microarrays (TMAs) with TRABD2B Antibody, HRP conjugated, several methodological considerations are critical:

• TMA Design and Sample Selection:

  • Include tissues representing different cancer stages and grades

  • Incorporate matched normal adjacent tissue controls for each patient

  • Consider inclusion of tissues known to have altered Wnt signaling

  • For renal cell carcinoma and osteosarcoma studies, include samples from different subtypes to account for molecular heterogeneity

• Staining Protocol Optimization:

  • Perform antigen retrieval optimization (test pH 6.0 citrate and pH 9.0 EDTA buffers)

  • Titrate antibody concentration (start with 1:100 to 1:500 dilutions)

  • Use automated staining platforms when possible to ensure consistency

  • Include positive control tissues (based on TRABD2B expression data in Human Protein Atlas)

• Scoring and Analysis Methodology:

  • Develop a standardized scoring system (e.g., H-score = intensity × percentage)

  • Employ digital pathology and automated image analysis for objective quantification

  • Have multiple pathologists score independently to establish inter-observer reliability

  • Correlate TRABD2B expression with established biomarkers and clinical outcomes

• Validation Strategy:

  • Confirm TMA findings on whole tissue sections from a subset of cases

  • Validate protein expression with orthogonal methods (RNA-ISH, RT-qPCR from matched samples)

  • Perform statistical analysis accounting for multiple comparisons and potential confounding variables

• Data Integration Approach:

  • Integrate TRABD2B expression data with genomic and clinical information

  • Perform multivariate analysis to assess independent prognostic value

  • Consider consensus molecular subtypes of the cancer type being studied

  • Analyze correlation with Wnt pathway activation markers

This comprehensive methodological approach will generate reliable data on TRABD2B's role in cancer progression that can be validated across patient cohorts .

How can researchers design experiments to investigate the metal ion dependency of TRABD2B's enzymatic activity?

To investigate the metal ion dependency of TRABD2B's enzymatic activity, researchers can design experiments utilizing TRABD2B Antibody, HRP conjugated in the following comprehensive approach:

• Enzyme Purification and Activity Assay Development:

  • Immunoprecipitate TRABD2B from cell lysates using the antibody

  • Develop a fluorogenic or chromogenic peptide substrate based on known Wnt cleavage sites

  • Establish baseline activity measurements in standard buffer conditions

• Metal Ion Dependency Profiling:

  • Systematically test enzyme activity across a panel of divalent cations:

    • Known cofactors: Mn²⁺, Co²⁺

    • Related metals: Zn²⁺, Fe²⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺

  • Create a concentration-response curve for each metal (0.1-10 mM)

  • Measure activity in terms of Vmax and Km for each condition

• Chelation Studies:

  • Assess activity inhibition patterns with different chelators:

    • EDTA (broad spectrum)

    • EGTA (Ca²⁺-selective)

    • 1,10-phenanthroline (Zn²⁺-selective)

  • Determine IC₅₀ values for each chelator

  • Test reversibility by metal ion replenishment after chelation

• Structural Analysis Approaches:

  • Perform site-directed mutagenesis of predicted metal-coordinating residues

  • Measure activity of mutants in the presence of different metal ions

  • Use circular dichroism to assess structural changes in TRABD2B upon metal binding

  • Consider computational modeling of the metalloprotease domain

• Cellular Validation:

  • Manipulate cellular metal ion availability using cell-permeable chelators

  • Monitor TRABD2B-dependent Wnt processing in these conditions

  • Correlate with functional Wnt signaling outputs (using TOPFlash assays)

  • Assess changes in TRABD2B subcellular localization in response to metal availability