RELT Antibody

What is RELT and why is it significant in scientific research?

RELT (Receptor Expressed in Lymphoid Tissues) is a type I transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily (TNFRSF), specifically designated as TNFRSF19L. Human RELT consists of 430 amino acid residues with a putative 26 amino acid signal peptide, a 136 amino acid extracellular domain containing one TNF receptor cysteine-rich domain, a 21 amino acid transmembrane domain, and a 247 amino acid cytoplasmic region without a death domain .

RELT is primarily expressed in hematopoietic tissues and peripheral blood leukocytes. Its significance in research stems from its roles in immune response regulation, as it has been shown to exclusively bind the adaptor protein TNF receptor-associated factor 1 (TRAF1) and activate the NF-kappa B pathway independently of TRAFs. Additionally, immobilized RELT can co-stimulate T-cell proliferation in the presence of CD3 signaling, suggesting a potential regulatory role in immune responses .

Recent studies have also associated RELT with various pathological conditions, including B-cell lymphomas, gastric cancer, breast cancer, and lung cancer, making it an important target for immunological and oncological research .

What are the main applications for RELT antibodies in laboratory research?

RELT antibodies have several key applications in scientific research:

• Western Blotting: RELT antibodies can detect RELT protein in human tissue lysates such as bone marrow and lymph node tissues, typically revealing a specific band at approximately 50 kDa .

• Flow Cytometry: Monoclonal RELT antibodies can be used to detect RELT expression in cell lines such as Raji human Burkitt's lymphoma cells .

• Immunohistochemistry: RELT antibodies have been employed to visualize RELT expression in normal human lymph nodes and B-cell lymphomas, where they've revealed differential expression patterns .

• ELISA: Direct ELISA applications have been documented for detecting recombinant human RELT protein .

• Co-immunoprecipitation: RELT antibodies have been used in co-IP studies to investigate protein-protein interactions, such as those between RELT and MDFIC .

It's important to note that optimal dilutions for each application should be determined by individual laboratories, as efficiency may vary based on experimental conditions and antibody lots .

What controls should be included when using RELT antibodies in experimental protocols?

For rigorous experimental design with RELT antibodies, the following controls should be implemented:

• Negative Controls:

  • Isotype controls: Use matched isotype antibodies (e.g., MAB0041 for mouse monoclonal anti-RELT antibodies) to assess non-specific binding .

  • Knockout (KO) cell lines: These represent the gold standard negative control, particularly for Western blot and immunofluorescence applications. The YCharOS group demonstrated that KO cell lines are superior to other control types for antibody validation .

  • Secondary antibody-only controls: To evaluate background from secondary detection reagents.

• Positive Controls:

  • Known RELT-expressing tissues/cells: Human lymph node tissue, bone marrow, and Raji cells have been documented to express RELT .

  • Recombinant RELT protein: Can serve as a standard for detection specificity.

• Specificity Controls:

  • Cross-reactivity assessment: Test against related proteins such as other TNFRSF members or RELL1/RELL2 .

  • Blocking peptides: Pre-incubation with the immunizing peptide should abolish specific staining.

• Loading/Technical Controls:

  • Housekeeping proteins (for Western blot)

  • Buffer-only samples

  • Multiple antibody lots when possible to address lot-to-lot variation

The YCharOS study emphasized that approximately 12 publications per protein target included data from antibodies that failed to recognize the relevant target protein, highlighting the critical importance of proper controls .

How can researchers optimize RELT antibody-based Western blotting protocols?

For optimal Western blot detection of RELT, researchers should consider the following methodological approaches:

• Sample Preparation:

  • Use appropriate lysis buffers compatible with membrane proteins

  • Include protease inhibitors to prevent degradation

  • For RELT detection, both human bone marrow and lymph node tissues have been successfully used as positive control samples

• Running Conditions:

  • Use reducing conditions as documented in successful RELT Western blots

  • PVDF membrane appears to work well for RELT detection

  • A 50 kDa band is the expected size for RELT detection

• Antibody Concentrations and Incubation:

  • Start with 1 μg/mL concentration for primary antibody (based on successful detection in published protocols)

  • For secondary detection, HRP-conjugated anti-species antibodies have shown good results

• Buffer Selection:

  • Immunoblot Buffer Group 1 has been documented to work well with RELT antibodies

• Signal Enhancement and Background Reduction:

  • Consider using highly sensitive ECL substrates for detection

  • Optimize blocking conditions to reduce non-specific binding

  • Multiple washing steps are crucial for reducing background

• Validation Approach:

  • Confirm results with multiple antibodies targeting different epitopes of RELT when possible

  • Include knockout or knockdown controls to verify specificity

By meticulously optimizing these parameters, researchers can achieve reliable and reproducible Western blot results for RELT detection.

What are best practices for using RELT antibodies in immunohistochemistry of lymphoid tissues?

Based on published research using RELT antibodies in immunohistochemistry of lymphoid tissues, the following best practices are recommended:

• Tissue Processing and Fixation:

  • Formalin-fixed, paraffin-embedded sections of lymphoid tissues have been successfully used for RELT IHC

  • Consider optimization of antigen retrieval methods, as TNFRSF members may be sensitive to fixation

• Staining Protocol Development:

  • Include both normal lymph nodes and B-cell lymphoma tissues as controls

  • RELT staining has been observed to be most prominent in macrophages in normal lymph nodes

  • In B-cell lymphomas, co-staining with CD20 has been used to confirm RELT expression in malignant B cells

• Quantification and Analysis:

  • Semi-quantitative measurement of RELT expression can be performed, as demonstrated in published studies

  • The following data has been reported for RELT staining intensity (mean ± SEM, N=8) :

    Sample	RELT staining intensity
    Normal Lymph Node	53.8 ± 19.3
    Low-Grade B-cell Lymphoma	81.3 ± 18.2
    High-Grade B-cell Lymphoma	185 ± 34.6

• Multiplexed Analysis:

  • Consider dual immunofluorescence with lineage markers (CD20, CD3, CD68) to identify cell types expressing RELT

  • This approach has successfully demonstrated progressive increase in RELT expression from normal lymphoid tissue to high-grade B-cell lymphomas

• Controls and Validation:

  • Use statistical analysis (e.g., one-way ANOVA) to evaluate staining differences between sample groups

  • Include isotype controls and secondary-only controls

  • When possible, validate IHC findings with other methods such as flow cytometry or Western blotting

These methodological approaches should help researchers obtain reliable and reproducible RELT staining patterns in lymphoid tissues.

How does RELT expression differ between normal lymphoid tissue and B-cell lymphomas?

RELT expression shows significant differential patterns between normal lymphoid tissues and B-cell lymphomas, which has important implications for both diagnostic and research applications:

• Expression Level Differences:

  • Immunohistochemical studies have demonstrated a progressive increase in RELT staining intensity from normal lymph nodes to high-grade B-cell lymphomas

  • Quantitative analysis revealed the following staining intensity values (mean ± SEM, N=8) :

    • Normal Lymph Node: 53.8 ± 19.3

    • Low-Grade B-cell Lymphoma: 81.3 ± 18.2

    • High-Grade B-cell Lymphoma: 185 ± 34.6

  • Statistical analysis confirmed these differences are significant (p < 0.05)

• Cellular Distribution Patterns:

  • In normal lymph nodes, RELT staining is most prominent in macrophages

  • In B-cell lymphomas, co-staining with CD20 reveals RELT expression in malignant B cells, suggesting a shift in expression pattern

  • This indicates that RELT expression may be upregulated in malignant B cells compared to normal B cells

• Research Implications:

  • The progressive increase in RELT expression correlating with lymphoma grade suggests RELT may play a role in B-cell lymphoma pathogenesis or progression

  • These findings align with other cancer studies that have identified RELT upregulation in various malignancies, including gastric cancer and lung cancer

  • The distinct expression patterns could potentially be exploited for diagnostic purposes or as therapeutic targets

• Methodological Considerations:

  • When studying RELT in lymphoma contexts, researchers should include both normal and malignant tissues for comparison

  • Multiple-marker approaches (co-staining) are essential to accurately identify the cell types expressing RELT

This differential expression pattern makes RELT a potentially valuable marker for studying B-cell lymphoma biology and may have future implications for lymphoma diagnosis or treatment strategies.

What are the known protein interactions of RELT and how do they influence antibody selection?

RELT participates in several protein-protein interactions that have significant implications for antibody selection and experimental design:

• TRAF1 Interaction:

  • RELT has been shown to exclusively bind the adaptor protein TNF receptor-associated factor 1 (TRAF1)

  • This interaction mediates signaling downstream of RELT

  • Antibodies targeting epitopes involved in TRAF1 binding may interfere with this interaction, which could be either desirable or problematic depending on the research question

• MDFIC Association:

  • The transcription factor MyoD family inhibitor domain-containing (MDFIC) was identified as a binding partner for RELT family members

  • MDFIC co-localizes with RELT at the plasma membrane

  • When studying this interaction, antibodies should be selected to avoid epitopes at the MDFIC binding interface

• RELL1 and RELL2 Homologs:

  • RELL1 and RELL2 are homologs that physically interact with RELT and co-localize at the plasma membrane

  • These proteins share structural features with RELT

  • Antibody selection must consider potential cross-reactivity with these homologs

  • For specific detection of RELT versus RELL1/RELL2, epitopes unique to RELT should be targeted

• Structural Considerations:

  • Computational analysis has revealed that RELT family members are highly disordered proteins

  • This structural characteristic may affect epitope accessibility and antibody binding efficacy

  • Antibodies targeting more structured regions may provide more consistent results

• Recommendations for Antibody Selection:

  • For studies of RELT signaling pathways, select antibodies that don't interfere with known protein interaction sites

  • For co-immunoprecipitation studies, choose antibodies validated for this application that don't compete with binding partners

  • When differential detection of RELT versus RELL homologs is important, select antibodies with demonstrated lack of cross-reactivity

  • Consider using antibodies targeting the extracellular domain for cell surface detection, and cytoplasmic domain antibodies for intracellular signaling studies

Understanding these protein interactions helps researchers select appropriate antibodies that won't interfere with the biological processes under investigation or lead to confounding results due to cross-reactivity issues.

What challenges exist in developing highly specific RELT antibodies?

Developing highly specific RELT antibodies presents several significant challenges that researchers and antibody manufacturers must address:

• Structural Complexity and Disorder:

  • Computational analysis has revealed that RELT family members are highly disordered proteins

  • Disordered regions may adopt different conformations in different contexts, potentially affecting epitope presentation and antibody recognition

  • This structural flexibility makes it difficult to predict which epitopes will be consistently accessible for antibody binding

• Homology with Related Proteins:

  • RELT shares structural similarities with other TNFRSF members, particularly TNFRSF19 and OX40

  • RELT also has homologs RELL1 and RELL2 that share sequence homology

  • These similarities increase the risk of cross-reactivity, requiring careful epitope selection and extensive validation

• Post-translational Modifications:

  • RELT undergoes post-translational modifications including phosphorylation and glycosylation

  • The presence of a potential N-linked glycosylation site in the extracellular domain means that antibodies targeting this region must recognize the protein regardless of glycosylation state

  • These modifications may mask epitopes or create conformational changes affecting antibody binding

• Validation Challenges:

  • The need for appropriate negative controls (knockout cell lines) has been emphasized in recent literature as critical for proper antibody validation

  • Approximately 12 publications per protein target have included data from antibodies that failed to recognize their target , highlighting the scope of the problem

  • YCharOS testing revealed that vendors needed to remove ~20% of tested antibodies that failed to meet expectations

• Reproducibility Issues:

  • Lot-to-lot variation remains a significant challenge in antibody production

  • The Research Resource Identifier (RRID) program highlights that different lots of the same manufacturer's antibody share the same RRID, even though lot-to-lot variation may exist

  • This variability necessitates repeated validation for each new lot

• Solutions and Best Practices:

  • Recombinant antibody technology offers a promising approach, as recombinant antibodies have been shown to outperform both monoclonal and polyclonal antibodies across multiple assays

  • Multi-epitope targeting approach, using antibodies against different regions of RELT

  • Rigorous validation across multiple applications using knockout controls

  • Detailed documentation of the specific epitope targeted and validation methods used

These challenges underscore the importance of stringent validation protocols and the potential advantages of recombinant antibody technology for developing reliable RELT-specific reagents.

How should researchers properly document and cite RELT antibodies in publications?

Proper documentation and citation of RELT antibodies in scientific publications is crucial for experimental reproducibility. The following best practices should be implemented:

• Essential Information to Include:

  • Antibody name with target (e.g., "Anti-Human RELT/TNFRSF19L Antibody")

  • Clone number for monoclonal antibodies (e.g., "Clone # 238104")

  • Manufacturer/vendor name and catalog number (e.g., "R&D Systems, Catalog # MAB1385")

  • Antibody type (monoclonal/polyclonal, host species, isotype)

  • Target epitope information when available (e.g., "Met1-Ala160 (Arg127Gly, Arg129Gly)")

  • Lot number (crucial due to lot-to-lot variations)

  • Working dilution used for each specific application

  • RRID (Research Resource Identifier) number

• RRID Implementation:

  • The Research Resource Identifier (RRID) program generates unique identifiers for antibodies and other reagents

  • Including RRIDs has seen steady increases in use, with >5,000 articles in >380 journals including RRID data by 2017

  • RRIDs allow tracking antibody use across the literature

  • Example format: "Anti-Human RELT/TNFRSF19L Antibody (R&D Systems, Catalog # MAB1385, RRID:AB_123456)"

• Methods Section Reporting:

  • Describe validation steps performed (e.g., testing on knockout controls)

  • Include information about controls used in the experiment

  • Detail any modifications to standard protocols

  • Specify application-specific conditions (e.g., "for Western blot, the antibody was used at 1 μg/mL under reducing conditions")

• Addressing Reproducibility Concerns:

  • The Antibody Registry has significantly impacted antibody reagent identifiability in the literature, with over 300,000 RRIDs for antibodies used across 46,500 papers and 2,000 journals

  • A study of accessible antibody sentences in open access literature found unique identifiability of antibodies increasing from 12% in 1997 to 31% in 2020

  • Tools like SciScore can help identify the presence or lack of important identifying information

• Data Repository Considerations:

  • When possible, deposit raw data and detailed protocols in appropriate repositories

  • Consider sharing detailed antibody validation data through platforms that connect to the RRID ecosystem

Proper documentation not only enhances reproducibility but also contributes to the collective knowledge base regarding antibody performance, potentially saving other researchers time and resources in their experimental design.

What strategies can address lot-to-lot variation in RELT antibodies?

Lot-to-lot variation represents a significant challenge to experimental reproducibility when working with RELT antibodies. Researchers can implement several strategies to mitigate this issue:

• Comprehensive Lot-Specific Validation:

  • Each new antibody lot should undergo validation using the specific application(s) intended for the study

  • Knockout or knockdown controls are the gold standard for specificity validation

  • Compare new lots directly against previous lots that performed well

  • Document lot-specific optimal working concentrations and conditions

• Strategic Antibody Purchasing:

  • Purchase sufficient quantity of a validated lot for completion of an entire study when possible

  • Consider requesting certificate of analysis with lot-specific validation data from vendors

  • For critical studies, request retention or reservation of specific lots that have been validated

• Recombinant Antibody Adoption:

  • The YCharOS study demonstrated that recombinant antibodies outperformed both monoclonal and polyclonal antibodies across multiple assays

  • Recombinant antibodies offer greater lot-to-lot consistency due to their defined sequence and production method

  • While current commercial catalogs may not offer recombinant anti-RELT antibodies, this represents a promising future direction

• Control Implementation Strategies:

  • Maintain frozen aliquots of positive control samples (e.g., RELT-expressing cell lysates) to test each new antibody lot

  • Include internal controls in each experiment that allow normalization between different antibody lots

  • Run side-by-side comparisons when transitioning between lots

• Documentation and Reporting Practices:

  • Always report lot numbers in laboratory notebooks and publications

  • Document observed differences between lots for internal reference

  • Consider publishing lot-specific validation data in supplementary materials

• Community Solutions:

  • Contribute to antibody validation databases that track lot-specific performance

  • Share validation data through platforms connected to RRID

  • Support initiatives like YCharOS that perform independent antibody characterization

By implementing these strategies, researchers can better manage the inherent variability in antibody reagents and improve the reliability and reproducibility of RELT-related research findings.

How might RELT antibodies be utilized in studying B-cell lymphoma pathogenesis?

Given the observed upregulation of RELT in B-cell lymphomas, RELT antibodies offer promising tools for investigating lymphoma biology and potential therapeutic applications:

• Diagnostic and Prognostic Applications:

  • The progressive increase in RELT expression from normal lymph nodes (53.8 ± 19.3) to low-grade (81.3 ± 18.2) and high-grade (185 ± 34.6) B-cell lymphomas suggests potential value as a disease progression marker

  • RELT antibodies could be developed into immunohistochemical tools for lymphoma subtyping or prognosis prediction

  • Multiplexed approaches combining RELT with established lymphoma markers might enhance diagnostic precision

• Mechanistic Studies:

  • RELT antibodies can help elucidate whether RELT overexpression is a driver or consequence of B-cell malignant transformation

  • Investigation of RELT signaling in lymphoma cells using co-immunoprecipitation with validated antibodies could reveal lymphoma-specific interaction partners

  • The relationship between RELT and the NF-kappa B pathway (known to be dysregulated in many B-cell lymphomas) could be explored using specific antibodies

• Functional Studies:

  • Blocking antibodies against RELT could be developed to determine if RELT signaling is essential for lymphoma cell survival or proliferation

  • Flow cytometry with RELT antibodies could be used to isolate RELT-high versus RELT-low populations for comparative functional studies

  • CRISPR-engineered cell lines with RELT knockout or mutation could be used alongside RELT antibodies to validate findings

• Therapeutic Development:

  • If RELT is confirmed as functionally important in lymphoma biology, antibody-based therapeutics might be developed

  • Combined approaches using RELT-targeted antibodies with established lymphoma therapies could be investigated

  • Antibody-drug conjugates targeting RELT might offer selective delivery of cytotoxic agents to lymphoma cells

• Research Methodology Considerations:

  • RELT antibodies with different epitope specificities should be employed to ensure comprehensive detection of potential isoforms or post-translationally modified versions

  • Quantitative approaches (such as multiplex IHC or mass cytometry) using RELT antibodies could reveal heterogeneity within lymphoma populations

  • Studies correlating RELT expression with clinical outcomes would benefit from standardized RELT antibody-based detection methods

This research direction highlights how well-characterized RELT antibodies could significantly advance our understanding of B-cell lymphoma biology and potentially lead to new diagnostic or therapeutic strategies.

What advances in antibody design might improve future RELT antibody development?

Recent technological advances in antibody engineering and characterization promise to enhance the development of next-generation RELT antibodies:

• Deep Learning and Linear Programming Approaches:

  • Novel antibody library design methods now combine deep learning and multi-objective linear programming with diversity constraints

  • These computational approaches leverage sequence and structure-based deep learning to predict mutation effects on antibody properties

  • Applied to RELT antibodies, such methods could generate diverse libraries with optimized binding properties without requiring extensive wet lab experimentation

• Recombinant Antibody Technology:

  • Studies have demonstrated that recombinant antibodies consistently outperform both monoclonal and polyclonal antibodies across multiple assays

  • For RELT detection, recombinant antibodies would offer several advantages:

    • Elimination of batch-to-batch variability

    • Renewable supply of identical antibodies

    • Potential for engineering to enhance specificity for RELT over related proteins

• Structural Biology Integration:

  • Considering that RELT family members are highly disordered proteins , structure-guided antibody design could target more ordered regions

  • Computational prediction of epitope accessibility in disordered regions

  • Design of antibodies that can recognize RELT regardless of its conformational state

• Multi-Parameter Optimization:

  • Modern antibody design can simultaneously optimize for multiple parameters including:

    • Specificity for RELT over related TNFRSF members

    • Performance across multiple applications (Western blot, IHC, flow cytometry)

    • Stability and storage characteristics

  • This approach could yield versatile RELT antibodies suitable for various research contexts

• Validation Technology:

  • Knockout cell line validation has proven superior for antibody testing

  • High-throughput validation platforms using CRISPR-modified cell lines expressing varying levels of RELT could accelerate antibody screening

  • Standardized validation pipelines following the YCharOS model could objectively compare different RELT antibodies

• Community-Driven Initiatives:

  • Open science approaches like the Only Good Antibodies (OGA) community could facilitate sharing of RELT antibody characterization data

  • Consortia focusing on TNFRSF member antibodies could develop standardized validation protocols specific to this receptor family

  • These collaborative approaches would accelerate the identification of optimal RELT antibody reagents

These advances suggest that future RELT antibodies will likely offer superior specificity, reproducibility, and application versatility compared to current reagents, enhancing the quality and reliability of RELT-focused research.

What are the key considerations for researchers selecting RELT antibodies for their studies?

When selecting RELT antibodies for research purposes, investigators should consider the following key factors to ensure experimental success and data reliability:

• Experimental Application Compatibility:

  • Different applications require antibodies with specific properties (e.g., recognition of native vs. denatured protein)

  • Verify that the antibody has been validated specifically for your intended application (Western blot, IHC, flow cytometry, etc.)

  • Review scientific data showing performance in your application of interest, such as Western blot data showing the expected 50 kDa band for RELT

• Specificity and Validation Status:

  • Prioritize antibodies tested against knockout controls, which provide the gold standard for specificity validation

  • Check for potential cross-reactivity with other TNFRSF members or RELL1/RELL2 homologs

  • Review vendor validation data and independent validation studies when available

  • Consider that approximately 20% of commercially available antibodies failed to meet expectations in systematic testing

• Antibody Format and Production Method:

  • Recombinant antibodies have demonstrated superior performance across multiple assays compared to monoclonal and polyclonal antibodies

  • Monoclonal antibodies offer consistency but may recognize limited epitopes

  • Polyclonal antibodies recognize multiple epitopes but have greater batch-to-batch variation

  • Consider the antibody's host species when planning multiplexed experiments

• Epitope Characteristics:

  • Consider whether the epitope is in the extracellular domain (aa 26-160) or intracellular domain of RELT

  • Be aware that RELT family members are highly disordered proteins , which may affect epitope accessibility

  • Check if the epitope includes or is affected by post-translational modifications like glycosylation

• Documentation and Reproducibility Factors:

  • Select antibodies with Research Resource Identifiers (RRIDs) for proper citation and tracking

  • Consider lot-to-lot consistency issues and whether critical experiments can be completed with a single lot

  • Review published literature using the antibody to assess its performance in peer-reviewed research

• Experimental Design Considerations:

  • Plan for appropriate positive and negative controls based on known RELT expression patterns

  • Consider the differential expression of RELT in normal tissues versus B-cell lymphomas when selecting control samples

  • Factor in storage requirements and stability information when planning long-term studies

By carefully evaluating these factors, researchers can select RELT antibodies most likely to provide reliable, reproducible results for their specific experimental needs, ultimately enhancing the quality and impact of their research.