TRABD2B Antibody

What is TRABD2B and what is its biological function?

TRABD2B, also known as Tiki2 or HKAT, is a 517 amino acid metalloproteinase that functions as a negative regulator of Wnt signaling. It mediates the cleavage of N-terminal residues in many Wnt proteins, leading to their inactivation. This protein is dependent on metal ions (specifically Mn²⁺/Co²⁺) for its activity and can be inhibited by divalent metal chelators such as EDTA. TRABD2B has been found to be upregulated in renal cell carcinoma and appears to suppress the growth of osteosarcoma by targeting the canonical Wnt pathway .

Unlike many other metalloproteases, the Tiki family proteins (including TRABD2B) represent a unique class of enzymes with specific functions in developmental and oncological contexts. Their activity in cleaving and subsequently causing oxidation of Wnt proteins creates a distinctive regulatory mechanism in this crucial signaling pathway .

How does TRABD2B/Tiki2 differ from TRABD2A/Tiki1?

While both TRABD2B (Tiki2) and TRABD2A (Tiki1) function as negative regulators of Wnt signaling through cleavage of Wnt proteins, they have several important differences:

TRABD2A/Tiki1 was initially identified through functional screening as an organizer-specific protein required for head formation in Xenopus. After cleaving the N-terminal residues from Wnt proteins, these proteins become oxidized and form large disulfide-bond oligomers, leading to their inactivation .

What are the optimal storage conditions for TRABD2B antibodies?

For maximum stability and activity retention, TRABD2B antibodies should be stored according to the following guidelines:

• Store antibodies at -20°C to -70°C for long-term storage (up to 12 months from receipt)

• For reconstituted antibodies, store at 2-8°C under sterile conditions for up to 1 month

• For longer storage after reconstitution, store at -20°C to -70°C for up to 6 months under sterile conditions

• Avoid repeated freeze-thaw cycles by aliquoting the antibody before freezing

• Most TRABD2B antibodies are supplied in a storage buffer containing PBS with preservatives such as 0.02% sodium azide and 50% glycerol at pH 7.3

The stability of antibodies can vary between manufacturers and formulations, so always check the product-specific storage recommendations. For example, some TRABD2B antibodies are stable for one year after shipment when stored at -20°C, and aliquoting may be unnecessary for -20°C storage in certain formulations .

What experimental validation methods should be used to confirm TRABD2B antibody specificity?

Ensuring antibody specificity is crucial for obtaining reliable research data. For TRABD2B antibodies, consider these validation approaches:

• Transfection-based validation: Compare signal between mock-transfected and TRABD2B-transfected cell lines (e.g., HEK293). This approach has been documented to show a specific band at approximately 65 kDa in Western blots of transfected cells .

• Multiple detection methods: Cross-validate using different techniques such as Western blot and flow cytometry on the same samples. For example, TRABD2B antibodies have been validated in both Western blot and flow cytometry applications using HEK293 cells transfected with human TRABD2B .

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide prior to application to demonstrate signal reduction.

• Knockdown/knockout validation: Compare signal in wild-type cells versus those with TRABD2B knockdown/knockout to confirm specificity.

• Cross-reactivity testing: If your research involves multiple species, test the antibody against samples from all relevant species to confirm reactivity. Current commercially available antibodies primarily show reactivity with human samples .

• Multiple antibody comparison: Use antibodies from different manufacturers or those targeting different epitopes of TRABD2B to confirm consistency in results.

How can discrepancies between calculated and observed molecular weights of TRABD2B be explained?

The calculated molecular weight of TRABD2B is approximately 57 kDa, but the observed molecular weight in Western blot applications typically ranges from 65-79 kDa . This discrepancy can be explained by several factors:

• Post-translational modifications: As a metalloproteinase, TRABD2B likely undergoes various post-translational modifications including glycosylation, phosphorylation, or other modifications that increase its apparent molecular weight.

• Protein structure and SDS-binding: The tertiary structure of TRABD2B may affect SDS binding during SDS-PAGE, resulting in altered migration patterns.

• Experimental conditions: Different buffer systems and reducing conditions can affect protein migration. For example, documented Western blot detection of TRABD2B was conducted under reducing conditions using Immunoblot Buffer Group 1 .

• Isoforms and splicing variants: Though not extensively documented for TRABD2B, potential splice variants or isoforms could account for different observed molecular weights.

When analyzing Western blot results, it's important to note that the band corresponding to TRABD2B typically appears at approximately 65 kDa in HEK293 cells transfected with human TRABD2B , while other studies have observed bands in the 65-79 kDa range in various cell lines including DU 145, HT-29, and HeLa cells .

What is known about TRABD2B's role in cancer research, and how can antibodies help investigate this role?

TRABD2B has been implicated in various cancer types, making it an interesting target for oncology research:

• Upregulation in renal cell carcinoma: TRABD2B has been found to be upregulated in renal cell carcinoma, suggesting potential involvement in kidney cancer progression .

• Suppression of osteosarcoma growth: TRABD2B appears to suppress the growth of osteosarcoma by targeting the canonical Wnt pathway . This suggests a potential tumor suppressor role in bone cancer.

• Wnt pathway regulation: As a negative regulator of Wnt signaling, TRABD2B may influence cancer development and progression in multiple cancers where Wnt signaling is dysregulated.

TRABD2B antibodies can be utilized in cancer research through:

• Expression profiling: Comparing TRABD2B expression levels across normal and cancer tissues using immunohistochemistry or Western blotting.

• Functional studies: Evaluating the effects of TRABD2B overexpression or knockdown on cancer cell proliferation, migration, and invasion.

• Mechanistic investigations: Examining how TRABD2B affects Wnt pathway components and downstream targets in cancer cells.

• Biomarker potential: Assessing whether TRABD2B expression levels correlate with clinicopathological features or patient outcomes.

When investigating TRABD2B in cancer contexts, it's advisable to use multiple cell lines relevant to the cancer type of interest. Documented positive detections have been reported in cervical cancer (HeLa), prostate cancer (DU 145), and colorectal cancer (HT-29) cell lines .

What are the optimal protocols for detecting TRABD2B using Western blotting?

For successful Western blot detection of TRABD2B, follow these optimized protocols:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • For cell lines with endogenous expression, documented positive results have been obtained with DU 145, HT-29, and HeLa cells

  • For overexpression studies, HEK293 cells transfected with human TRABD2B provide good controls

• Gel electrophoresis and transfer:

  • Use PVDF membrane for optimal protein binding

  • Run samples under reducing conditions

  • Use Immunoblot Buffer Group 1 for consistent results

• Antibody dilutions and incubation:

  • Primary antibody: Use at 0.5-2.0 μg/mL depending on the specific antibody (polyclonal sheep anti-human TRABD2B has been used at 0.5 μg/mL , while rabbit polyclonal antibodies have been recommended at 1:500-1:1000 dilution )

  • Secondary antibody: Use appropriate HRP-conjugated secondary antibody (e.g., HRP-conjugated Anti-Sheep IgG or Anti-Rabbit IgG)

  • Expected band: Look for a specific band at approximately 65-79 kDa

• Controls:

  • Positive control: Lysate from TRABD2B-transfected cells

  • Negative control: Mock-transfected cells or irrelevant transfectants

• Optimization notes:

  • It is recommended that optimal dilutions be determined by each laboratory for each application

  • Results may be sample-dependent, so checking validation data is advisable

How can TRABD2B be effectively detected using flow cytometry?

Flow cytometry can be an effective method for detecting TRABD2B expression, particularly in transfected cell models:

• Sample preparation:

  • Prepare single-cell suspensions from cultured cells

  • HEK293 cells transfected with human TRABD2B provide good positive controls

• Staining protocol:

  • Use sheep anti-human TRABD2B antigen affinity-purified polyclonal antibody as primary antibody

  • Follow with fluorophore-conjugated secondary antibody (e.g., Allophycocyanin-conjugated Anti-Sheep IgG)

  • Alternatively, use directly conjugated antibodies such as FITC-conjugated TRABD2B antibodies for single-step staining

• Controls:

  • Positive control: Cells transfected with human TRABD2B

  • Negative control: Irrelevant transfectants or mock-transfected cells

  • Isotype control: Appropriate IgG matching the primary antibody's host species

• Analysis considerations:

  • Compare filled histogram (TRABD2B-transfected cells) versus open histogram (irrelevant transfectants) to confirm specific staining

  • Follow established protocols for staining membrane-associated proteins

• Troubleshooting:

  • If signal is weak, consider permeabilization to detect intracellular epitopes

  • Optimize antibody concentrations for your specific cell type

  • Ensure cells are viable and single-cell suspensions are achieved

What troubleshooting strategies can be applied when TRABD2B antibodies yield inconsistent results?

When facing inconsistent results with TRABD2B antibodies, consider these troubleshooting strategies:

• Antibody quality issues:

  • Check antibody expiration date and storage conditions

  • Avoid repeated freeze-thaw cycles by aliquoting antibodies

  • Consider testing antibodies from different lots or manufacturers

• Sample preparation problems:

  • Ensure complete protein denaturation for Western blotting

  • Add fresh protease inhibitors to lysis buffers

  • Optimize cell lysis conditions to ensure complete protein extraction

• Detection sensitivity:

  • For Western blot, increase protein loading (30-50 μg total protein)

  • Optimize antibody concentration (test a range from 0.1-2.0 μg/mL)

  • Increase incubation time or use more sensitive detection reagents

• Non-specific binding:

  • Increase blocking time or concentration of blocking agent

  • Use non-fat dry milk instead of BSA for blocking (or vice versa)

  • Add 0.1-0.3% Tween-20 to washing buffers to reduce background

• Epitope accessibility issues:

  • Try different fixation methods for immunohistochemistry or flow cytometry

  • For Western blotting, ensure complete protein denaturation

  • Consider native vs. reducing conditions if epitope is conformation-dependent

• Application-specific considerations:

  • For Western blot: Try both reducing and non-reducing conditions

  • For flow cytometry: Compare surface vs. intracellular staining protocols

  • For ELISA: Optimize coating concentration and blocking conditions

• Controls to implement:

  • Transfected vs. non-transfected cells as positive and negative controls

  • Include secondary antibody-only controls to check for non-specific binding

  • Use cell lines with documented TRABD2B expression (e.g., HeLa, DU 145, HT-29)

What new applications are emerging for TRABD2B antibodies in research?

As our understanding of TRABD2B's biological functions continues to evolve, several emerging applications for TRABD2B antibodies show promise:

• Cancer biomarker development: Given TRABD2B's differential expression in cancers like renal cell carcinoma and its suppressive role in osteosarcoma , antibodies may facilitate biomarker development for diagnostic or prognostic applications.

• Therapeutic target validation: TRABD2B antibodies can help validate this protein as a potential therapeutic target in Wnt-dependent cancers through immunoprecipitation and functional blocking studies.

• Developmental biology: As part of the Tiki family involved in head formation (like its homolog Tiki1) , TRABD2B antibodies may offer insights into developmental processes and congenital disorders.

• Live-cell imaging: Fluorescently conjugated antibodies like FITC-conjugated TRABD2B antibodies can be used for tracking protein dynamics in living cells with appropriate membrane permeabilization techniques.

• Single-cell analysis: Integration of TRABD2B antibodies into single-cell proteomics workflows could reveal heterogeneity in expression across different cell populations.

• Multiplex immunoassays: Incorporating TRABD2B detection into multiplexed platforms to simultaneously measure multiple components of the Wnt pathway.

• Structural biology: Using antibodies to stabilize protein conformations for cryo-EM or X-ray crystallography to better understand TRABD2B's metalloproteinase mechanism.

How might differential expression of TRABD2B across tissues impact experimental design?

TRABD2B (Tiki2) shows enriched expression in specific tissues including heart, kidney, and adipose tissue , which has important implications for experimental design:

• Tissue selection: When designing experiments to study endogenous TRABD2B, prioritize tissues with higher expression levels. For human samples, kidney tissues may be particularly relevant given the protein's upregulation in renal cell carcinoma .

• Cell line selection: Choose cell lines derived from tissues with known TRABD2B expression. Documented positive detection has been achieved in:

  • DU 145 (prostate cancer cells)

  • HT-29 (colorectal cancer cells)

  • HeLa (cervical cancer cells)

• Expression level considerations:

  • Low endogenous expression may require more sensitive detection methods

  • Consider using concentration steps (immunoprecipitation before Western blot)

  • Load higher amounts of total protein for tissues with lower expression

• Controls and normalization:

  • Include tissue-specific positive controls in experiments

  • Normalize expression to tissue-specific reference genes rather than universal housekeeping genes

  • Consider using transfected cell models (like TRABD2B-transfected HEK293 cells) as standardized positive controls

• Functional assays:

  • Design functional assays that account for tissue-specific interacting partners

  • Consider tissue-specific Wnt ligands when studying TRABD2B's role in Wnt signaling

  • Use tissue-relevant endpoints when assessing TRABD2B's biological effects

By accounting for tissue-specific expression patterns in experimental design, researchers can maximize the likelihood of detecting biologically relevant TRABD2B functions and avoid false negative results due to inappropriate tissue or cell line selection.

What are the best practices for antibody validation before conducting major TRABD2B studies?

Before undertaking extensive research projects involving TRABD2B antibodies, implementing a rigorous validation strategy is crucial:

• Multi-technique validation: Confirm antibody performance across multiple techniques relevant to your research (Western blot, immunofluorescence, flow cytometry, ELISA, etc.). Commercially available TRABD2B antibodies have documented performance in Western blot, flow cytometry, and ELISA applications .

• Positive and negative controls: Use appropriate controls including:

  • Overexpression systems (e.g., TRABD2B-transfected HEK293 cells)

  • Endogenous expression cell lines (DU 145, HT-29, HeLa)

  • Mock-transfected or irrelevant transfectants as negative controls

  • Knockdown/knockout models where possible

• Epitope mapping: Understand which region of TRABD2B your antibody targets. Some commercial antibodies target specific regions, such as aa 195-405 of the human TRABD2B protein .

• Lot-to-lot consistency testing: When purchasing new lots of the same antibody, perform side-by-side comparisons with previous lots to ensure consistent performance.

• Cross-reactivity assessment: Test for potential cross-reactivity with related proteins, particularly TRABD2A/Tiki1, which shares functional similarities with TRABD2B/Tiki2.

• Sample preparation optimization: Determine optimal fixation, permeabilization, and extraction methods for your specific samples and applications.

• Documentation: Maintain detailed records of validation experiments, including positive and negative results, to guide future studies and troubleshooting.

• Literature verification: Compare your validation results with published studies using the same or similar antibodies to ensure consistency with established findings.