TNFRSF9 Antibody, Biotin conjugated

What is TNFRSF9 and why is it a significant research target?

TNFRSF9 (Tumor Necrosis Factor Receptor Superfamily Member 9), also known as CD137 or 4-1BB, is a transmembrane protein belonging to the TNF receptor superfamily. It functions as a receptor for TNFSF9/4-1BBL and plays crucial roles in enhancing CD8+ T-cell survival, cytotoxicity, and mitochondrial activity, thereby promoting immunity against viruses and tumors. TNFRSF9 has gained significant attention as a therapeutic target for cancer and various autoimmune and inflammatory diseases due to its immunomodulatory properties . The 255 amino acid human 4-1BB contains four cysteine-rich motifs in its extracellular domain that are characteristic of the TNF receptor superfamily .

What are the expression patterns of TNFRSF9 in immune cells?

TNFRSF9 is absent from naive T cells but is upregulated and continually expressed following T cell activation. It is expressed on various activated immune cell populations including CD4+ T cells, CD8+ T cells, memory CD8+ T cells, NKT cells, regulatory T cells, dendritic cells, and mast cells . It can also be found on myeloid and mast cell progenitors, and even on bacterially infected osteoblasts . Understanding these expression patterns is crucial for properly designing experiments targeting specific cell types or activation states.

What is the significance of biotin conjugation for TNFRSF9 antibodies?

Biotin conjugation enables versatile detection methods through the strong affinity between biotin and streptavidin/avidin molecules. When TNFRSF9 antibodies are conjugated to biotin, they can be detected using various streptavidin-conjugated reporters such as Streptavidin-Allophycocyanin or NorthernLights™ 557-conjugated Streptavidin, as demonstrated in flow cytometry and immunocytochemistry applications . This conjugation allows for amplified signal detection, making it particularly valuable for detecting low-abundance proteins in complex biological samples.

What are the validated applications for biotin-conjugated TNFRSF9 antibodies?

Based on the search results, biotin-conjugated anti-TNFRSF9 antibodies have been validated for several applications:

• Western Blot (WB): Detection limit for recombinant human CD137 is 1.5-3.0 ng/lane under reducing or non-reducing conditions using 0.1-0.2 μg/ml antibody concentration .

• Enzyme-Linked Immunosorbent Assay (ELISA): Applicable at dilutions of 1:500-1000 .

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): Effective at dilutions of 1:200-400 .

• Flow Cytometry: Used to detect TNFRSF9 expression on activated T cells .

• Immunocytochemistry (ICC): Used to visualize TNFRSF9 in peripheral blood mononuclear cells .

How can TNFRSF9 antibodies be used in flow cytometry to identify activated T cell populations?

For flow cytometry applications, human T cells can be treated with activating agents (e.g., 5 μg/mL PHA for 48 hours) to induce TNFRSF9 expression. The cells are then stained with biotinylated anti-TNFRSF9 antibody followed by a streptavidin-conjugated fluorophore such as Streptavidin-Allophycocyanin. Using appropriate controls (such as irrelevant biotinylated antibodies of the same isotype), researchers can accurately identify TNFRSF9-expressing activated T cells . This approach allows for characterization of T cell activation status and can be combined with other markers to perform multiparameter analysis of immune cell populations.

What protocol is recommended for immunocytochemistry using biotin-conjugated TNFRSF9 antibodies?

For immunocytochemistry applications, the following protocol has been validated:

• Fix cells using an appropriate fixative (e.g., 4% paraformaldehyde).

• Apply biotinylated anti-TNFRSF9 antibody at a concentration of approximately 15 μg/mL.

• Incubate for 3 hours at room temperature.

• Detect using a streptavidin-conjugated fluorophore (e.g., NorthernLights™ 557-conjugated Streptavidin).

• Counterstain nuclei with DAPI.

• Visualize using fluorescence microscopy .

This protocol has been successfully used to detect TNFRSF9 in peripheral blood mononuclear cells, showing localization to both plasma membrane and cytoplasm.

How should Western blot conditions be optimized for detecting TNFRSF9 using biotinylated antibodies?

For optimal Western blot detection of TNFRSF9:

• Use antibody concentrations between 0.1-0.2 μg/ml.

• Both reducing and non-reducing conditions are suitable, with detection limits of 1.5-3.0 ng/lane for recombinant human CD137.

• Be aware that TNFRSF9 appears at multiple molecular weights: 24-28 kDa and 40-50 kDa bands may be observed due to post-translational modifications and dimerization .

• Include appropriate positive controls (recombinant TNFRSF9) and negative controls.

• For titration studies, serial dilutions of the target protein are recommended (e.g., 250, 125, 62.5, 31.25, 15.625, 7.8, 3.9, 1.95, 0.975, 0.4875, and 0.24 ng) .

What considerations are important when designing experiments to study TNFRSF9+ T cells in tumor microenvironments?

When studying TNFRSF9+ T cells in tumor contexts, researchers should consider:

These considerations should inform panel design for flow cytometry, selection of controls, and interpretation of results in tumor immunology studies.

How can researchers distinguish specific from non-specific staining when using biotinylated TNFRSF9 antibodies?

To distinguish specific from non-specific staining:

• Always include appropriate controls: use an irrelevant biotinylated antibody of the same isotype (e.g., BAF108 as shown in search result ).

• For flow cytometry, present data as filled histograms for specific staining and open histograms for control antibody staining .

• Validate results using multiple detection methods where possible (e.g., flow cytometry and immunocytochemistry).

• When possible, compare staining patterns between positive samples (e.g., activated T cells) and negative samples (e.g., resting T cells) to confirm specificity.

• For Western blots, include recombinant TNFRSF9 as a positive control to confirm antibody specificity and appropriate molecular weight .

What factors might affect the variability in TNFRSF9 detection across different experimental systems?

Several factors can influence variability in TNFRSF9 detection:

• Cell activation state: TNFRSF9 is expressed upon activation, so inconsistent activation protocols may lead to variable expression levels .

• Post-translational modifications: TNFRSF9 exists as both monomers and dimers on T cells and undergoes glycosylation, resulting in variable molecular weights (24-28 kDa and 40-50 kDa) .

• Sample preparation methods: Different fixation and permeabilization protocols may affect epitope accessibility.

• Antibody concentration: Optimal concentrations vary by application (0.1-0.2 μg/ml for WB; 15 μg/ml for ICC) .

• Detection systems: Different streptavidin conjugates may have varying sensitivity levels.

Researchers should standardize these variables and include appropriate controls to minimize experimental variability.

How can TNFRSF9 methylation status be assessed and what is its significance in immunotherapy research?

Analysis of TNFRSF9 DNA methylation has emerged as a potentially valuable biomarker for immunotherapy response:

This epigenetic approach offers a novel dimension for investigating TNFRSF9 biology beyond protein expression analysis.

What are the implications of TNFRSF9 bidirectional signaling for experimental design in cancer immunotherapy research?

The bidirectional signaling between TNFRSF9 and its ligand TNFSF9 introduces complexity that researchers must consider:

These complex interactions necessitate careful experimental design and comprehensive readouts when investigating TNFRSF9-targeted therapies.

What are the latest developments in TNFRSF9-targeted antibodies for cancer immunotherapy?

Recent developments in TNFRSF9-targeted immunotherapy include:

• Combination approaches: Studies have shown that combining TNFRSF9 agonists with PD-L1 inhibitors increases anti-tumor activity .

• Clinical trials: Multiple TNFRSF9-based antibodies are currently being tested in clinical trials for various cancers .

• Prediction of response: TNFRSF9+ CD8+ T cell infiltration patterns and TNFRSF9 methylation status may serve as biomarkers for response to immunotherapy, particularly checkpoint inhibitors like nivolumab .

• Mechanistic understanding: TNFRSF9 signals through the TFAF2-NIK pathway, resulting in activation of NF-kappa B and ultimately promoting proliferation and survival of T cells .

Researchers should consider these developments when designing studies involving TNFRSF9 targeting or using TNFRSF9 as a biomarker in immunotherapy contexts.

What are the key specifications to consider when selecting a biotin-conjugated TNFRSF9 antibody?

When selecting a biotin-conjugated TNFRSF9 antibody, researchers should consider:

• Host species and clonality: Options include rabbit polyclonal (e.g., bs-2449R-Biotin) and goat polyclonal (e.g., ab245844) antibodies .

• Immunogen: Check whether the antibody was raised against a recombinant fragment (e.g., human TNFRSF9 aa 1-200) or a synthetic peptide .

• Validated applications: Confirm that the antibody has been validated for your specific application (WB, ELISA, IHC-P, ICC, or flow cytometry) .

• Species reactivity: Verify reactivity with your species of interest (human, mouse, rat) .

• Storage conditions: Most antibodies require storage at -20°C in buffers containing glycerol and preservatives .

Careful consideration of these specifications will help ensure successful experimental outcomes.

How can researchers validate the functionality of biotin-conjugated TNFRSF9 antibodies before use in critical experiments?

To validate antibody functionality:

• Positive control experiments: Test the antibody on samples known to express TNFRSF9, such as PHA-stimulated T cells (5 μg/mL PHA for 48 hours) .

• Titration experiments: Perform antibody titrations to determine optimal concentrations for your specific application and sample type.

• Comparison with existing data: Compare staining patterns with published results showing TNFRSF9 localization to plasma membrane and cytoplasm in immune cells .

• Cross-validation: Use multiple detection methods (e.g., flow cytometry and Western blot) to confirm specificity.

• Blocking experiments: Pre-incubate the antibody with recombinant TNFRSF9 to demonstrate specific binding.

These validation steps are especially important for critical experiments or when using the antibody in novel applications or biological systems.

How is TNFRSF9 expression being used as a biomarker in cancer immunotherapy studies?

TNFRSF9 expression patterns are emerging as potential biomarkers in cancer immunotherapy: