TNFRSF1B Antibody

What is TNFRSF1B and why is it important in research?

TNFRSF1B (Tumor Necrosis Factor Receptor Superfamily, Member 1B), also known as TNF Receptor II (TNFR2), is one of two primary receptors for TNF-α. Unlike TNFR1 which is expressed ubiquitously, TNFRSF1B displays more restricted expression and plays crucial roles in immune regulation. It mediates immunomodulatory and neuroprotective activities, in contrast to TNFR1 which typically promotes pro-inflammatory and neurotoxic effects . TNFRSF1B is constitutively expressed at high levels on regulatory T cells (Tregs) and its expression correlates with their suppressive potential, making it a valuable marker and functional target in immunological research .

What are the key differences between TNFR1 (TNFRSF1A) and TNFR2 (TNFRSF1B) in terms of function?

In Alzheimer's disease research, TNFR1 protein levels and binding affinity are increased in AD brains, while TNFR2 levels and binding affinity are decreased compared to non-demented patients . This differential expression pattern correlates with their opposing functions in neuroinflammation.

What types of TNFRSF1B antibodies are commonly used in research?

Multiple types of TNFRSF1B antibodies are employed in research settings:

• Polyclonal antibodies: Recognize multiple epitopes on TNFRSF1B. Examples include rabbit polyclonal antibodies targeting specific regions such as:

  • Middle region antibodies (e.g., ABIN3044354 targeting AA 103-121)

  • C-terminal antibodies (e.g., ABIN3042335 targeting AA 288-318)

• Monoclonal antibodies: Recognize specific epitopes and provide consistent results. Examples include:

  • Mouse monoclonal antibody clone 22221 (MAB226)

  • Mouse monoclonal antibody clone 22235 (MAB2261)

• Functional antibodies: Include agonist antibodies that activate TNFRSF1B signaling and antagonist antibodies that block receptor function, particularly relevant in cancer research .

What are the validated applications for TNFRSF1B antibodies?

TNFRSF1B antibodies have been validated for multiple research applications:

For detection of human TNFRSF1B in flow cytometry, antibodies have been successfully used to identify expression on peripheral blood granulocytes and mononuclear cells .

How should I optimize western blotting protocols for TNFRSF1B detection?

Optimizing western blotting for TNFRSF1B requires attention to several specific parameters:

• Sample preparation:

  • Use lysis buffers containing protease inhibitors to prevent degradation

  • Include appropriate detergents (e.g., 1% Triton X-100) for membrane protein extraction

  • Avoid repeated freeze-thaw cycles of samples

• Gel electrophoresis:

  • Use 8-12% gels for optimal resolution of TNFRSF1B (approximately 48 kDa)

  • Include positive controls (e.g., cell lines known to express high levels of TNFRSF1B)

• Antibody conditions:

  • Recommended dilutions for TNFRSF1B antibodies range from 1:500 to 1:2,000

  • Consider overnight primary antibody incubation at 4°C for improved sensitivity

  • Test different blocking agents (5% non-fat milk vs. BSA) to determine optimal signal-to-noise ratio

• Troubleshooting multiple bands:

  • Multiple bands may represent glycosylated forms, soluble vs. membrane-bound forms, or degradation products

  • Use region-specific antibodies to help identify specific forms of TNFRSF1B

What considerations should be made when using TNFRSF1B antibodies for flow cytometry?

When using TNFRSF1B antibodies for flow cytometry, researchers should consider:

• Antibody selection and titration:

  • Choose antibodies specifically validated for flow cytometry (e.g., MAB226, MAB2261)

  • Titrate antibodies to determine optimal concentration (typically 0.25 μg per 10^6 cells for MAB226)

• Controls:

  • Include appropriate isotype controls (e.g., MAB003 as shown in search results)

  • Include unstained cells and single-color controls for compensation in multicolor panels

  • Consider TNFRSF1B knockout cells as negative controls when available

• Staining protocol optimization:

  • Use proper FcR blocking to reduce non-specific binding, especially for immune cells

  • Maintain cell viability throughout processing to avoid non-specific binding

  • For human peripheral blood, expect high expression on granulocytes and regulatory T cells

• Data analysis approaches:

  • Gate on viable cells to exclude dead cell autofluorescence

  • For quantitative analysis, consider using calibration beads to determine absolute receptor numbers

How can TNFRSF1B antibodies be used to study regulatory T cell function?

TNFRSF1B antibodies are valuable tools for studying regulatory T cell function due to the high constitutive expression of TNFRSF1B on Tregs:

• Identification of maximally suppressive Treg populations:

  • Use flow cytometry with anti-TNFRSF1B antibodies (e.g., MAB226) along with CD4, CD25, and FOXP3 markers

  • Human Tregs constitutively express high levels of TNFRSF1B relative to conventional T cells

  • TNFRSF1B expression correlates with the suppressive potential of natural Tregs

• Functional manipulation of Treg activity:

  • Agonistic anti-TNFRSF1B antibodies can enhance Treg function

  • Antagonistic antibodies may inhibit Treg-mediated immunosuppression

  • Combine with in vitro suppression assays to quantify functional impact

• Assessment of soluble TNFRSF1B production:

  • Activated Tregs release high amounts of soluble TNFRSF1B as an additional immunosuppressive mechanism

  • Measure soluble TNFRSF1B release using ELISA techniques

  • Evaluate the immunosuppressive activity of soluble TNFRSF1B in functional assays

What approaches are used to develop TNFRSF1B agonists versus antagonists for cancer immunotherapy?

The development of TNFRSF1B-targeting therapies involves distinct approaches for creating agonists versus antagonists:

Recent research has shown promising results:

• Tam et al. screened for anti-human and anti-mouse TNFRSF1B agonist antibodies and reported antitumor effects in several cancer types

• Enhanced efficacy was observed when combining TNFRSF1B agonists with anti-PD-1 therapy compared to PD-1 monotherapy

• Both agonists and antagonists targeting TNFRSF1B are being tested in the context of cancer therapy

How can TNFRSF1B antibodies be used to investigate TNF signaling in neuroinflammatory conditions?

TNFRSF1B antibodies are valuable tools for investigating the role of TNF signaling in neuroinflammatory conditions:

• Differential analysis of TNFR1 vs. TNFR2 signaling:

  • Use selective antibodies to distinguish between TNFR1 and TNFR2 expression in neural tissues

  • In Alzheimer's disease research, TNFR1 protein levels and binding affinity are increased in AD brains, while TNFR2 levels are decreased

  • TNFR1 signaling contributes to CNS neuroinflammation in transgenic AD mice and upon AβO injection

• Functional studies in neuroinflammation models:

  • Antagonistic TNFRSF1B antibodies can help determine the neuroprotective role of TNFR2 signaling

  • Evaluate how TNFR2 inhibition affects Aβ toxicity in vitro

  • Assess the impact of TNFR1/TNFR2 balance on neuroinflammatory processes

• Therapeutic targeting strategies:

  • Counteracting the effects of TNFR1 has therapeutic potential in neuroinflammatory conditions

  • TNFRSF1B antibodies can help identify cell populations that would benefit from selective targeting

  • Evaluate the effects of TNFR2 activation on neuroprotection versus immunomodulation

What are common issues when using TNFRSF1B antibodies in immunohistochemistry?

When using TNFRSF1B antibodies for immunohistochemistry, researchers may encounter several challenges:

• Antigen retrieval optimization:

  • TNFRSF1B is a membrane protein that may require specialized antigen retrieval methods

  • Test different retrieval conditions (pH, buffer composition, temperature, duration)

  • Compare heat-induced vs. enzyme-induced antigen retrieval methods

• Signal intensity issues:

  • Low expression levels in certain tissues may require signal amplification

  • Optimize antibody concentration (typically 1:50-1:200 dilution for IHC)

  • Extended primary antibody incubation (overnight at 4°C) may improve signal

• Tissue-specific considerations:

  • Expression patterns differ between tissue types and cellular compartments

  • In kidney tissues, TNFRSF1B expression patterns differ between glomerular and tubulointerstitial regions

  • Consider cell-type specific markers for co-localization studies

How can I validate the specificity of TNFRSF1B antibodies?

Validating TNFRSF1B antibody specificity requires multiple complementary approaches:

• Knockout/knockdown controls:

  • Compare staining between wild-type and TNFRSF1B knockout samples

  • TNFRSF1B was detected in THP-1 cells but not in TNFRSF1B knockout THP-1 cells

  • Use siRNA or shRNA knockdown of TNFRSF1B as an alternative approach

• Peptide competition assays:

  • Pre-incubate the antibody with the immunizing peptide

  • For ABIN3044354, this would be the synthetic peptide corresponding to AA 103-121

  • For ABIN3042335, the peptide corresponding to AA 288-318

  • A specific antibody signal should be significantly reduced or eliminated

• Multiple antibody validation:

  • Use multiple antibodies targeting different epitopes of TNFRSF1B

  • Results should be consistent across different antibodies

  • Compare polyclonal and monoclonal antibody results

• Cross-reactivity testing:

  • Test antibody on samples from multiple species if claimed to be cross-reactive

  • Some antibodies are specific to human (ABIN3044354) , while others react with multiple species (A01437 reacts with human, mouse, and rat)

  • Verify absence of signal in tissues/cells known not to express TNFRSF1B

What controls should be included when evaluating TNFRSF1B expression in different cell types?

A comprehensive set of controls ensures reliable interpretation of TNFRSF1B expression analyses:

Specifically for flow cytometry, human peripheral blood granulocytes serve as excellent positive controls as they consistently express high levels of TNFRSF1B, as demonstrated in multiple studies .

How does TNF-α signaling through TNFRSF1B affect stem cell differentiation?

Recent research has revealed important roles for TNFRSF1B in regulating stem cell differentiation:

• Effects on chondrogenic differentiation:

  • TNF-α inhibits chondrogenic differentiation of human adipose stem cells (hADSCs) by activating RELA expression through TNFRSF1B

  • This pathway upregulates OPA1 expression, thereby increasing mitochondrial fusion

  • The TNFRSF1B-RELA-OPA1 axis represents a novel mechanism controlling stem cell fate

• Molecular mechanisms:

  • Gene microarray and RT-qPCR data showed that TNF-α exposure leads to increased expression of TNFRSF1B and RELA during chondrogenic differentiation of hADSCs

  • This signaling pathway affects mitochondrial dynamics, with prolonged and interconnected mitochondria observed during this process

• Research applications:

  • TNFRSF1B antibodies can help track receptor expression changes during differentiation

  • Functional blockade of TNFRSF1B signaling may be used to modulate stem cell fate decisions

  • These findings have implications for tissue engineering and regenerative medicine approaches

What is the role of TNFRSF1B in the context of Alzheimer's disease research?

TNFRSF1B plays a complex role in Alzheimer's disease pathology, with significant implications for therapeutic development:

• Expression patterns in AD:

  • TNFR2 (TNFRSF1B) levels and binding affinity are decreased in AD brains compared to non-demented patients

  • In contrast, TNFR1 levels and binding affinity are increased in AD brains

  • This altered balance may contribute to neuroinflammatory processes in AD

• Neuroprotective functions:

  • Inhibition of TNFR2 increases Aβ toxicity in vitro

  • TNFR2 signaling appears to have neuroprotective roles in contrast to the neurotoxic activities of TNFR1

  • These opposing functions suggest that selective targeting of these receptors could have therapeutic potential

• Research applications:

  • TNFRSF1B antibodies can be used to map receptor distribution in AD versus healthy brain tissues

  • Functional antibodies may help dissect the specific contributions of TNFR1 versus TNFR2 signaling to AD pathology

  • The development of TNFR2 agonists represents a potential therapeutic approach for neurodegenerative conditions