JAM2 Antibody

What is JAM2 and what biological functions does it serve?

JAM2 (junctional adhesion molecule 2) is a member of the immunoglobulin superfamily that plays critical roles in cell adhesion processes. It functions primarily at tight junctions between endothelial cells, forming continuous seals that serve as physical barriers preventing solutes and water from passing freely through paracellular spaces . JAM2 acts as an adhesive ligand for interactions with various immune cell types and is implicated in lymphocyte homing to secondary lymphoid organs . Additionally, research indicates its involvement in spermatid development . The protein is highly expressed in heart, placenta, lung, foreskin, and lymph node tissues, suggesting tissue-specific functions that may vary based on cellular context .

How do I select an appropriate JAM2 antibody for my research?

Selecting the appropriate JAM2 antibody requires consideration of several critical factors:

• Experimental application: Different applications demand specific antibody characteristics. For Western blotting, antibodies with high specificity for denatured epitopes are essential. For immunohistochemistry or immunofluorescence, antibodies that recognize native conformations are preferred .

• Species reactivity: JAM2 antibodies vary in their cross-reactivity profile. Some recognize only human JAM2, while others cross-react with mouse and rat orthologs . Match the antibody's reactivity to your experimental model organism.

• Clonality: Polyclonal antibodies (like ABIN2855842) offer broader epitope recognition, while monoclonal antibodies (such as clone 7E4C5 or 1G4) provide higher specificity for particular epitopes .

• Validation data: Prioritize antibodies with extensive validation for your specific application. For example, product ABIN7440674 has 10 validations across multiple applications, suggesting reliable performance .

What are the optimal dilutions for JAM2 antibodies in different applications?

Optimal dilutions vary based on the specific antibody and application:

It is strongly recommended to titrate each antibody in your specific experimental system to achieve optimal signal-to-noise ratio . Different tissue sources and sample preparation methods may necessitate adjustment of these recommended ranges.

How should I validate the specificity of a JAM2 antibody?

Rigorous validation of JAM2 antibody specificity requires a multi-faceted approach:

• Positive and negative controls: Utilize tissues/cells known to express high levels of JAM2 (e.g., heart, placenta) as positive controls and tissues with minimal expression as negative controls .

• Knockdown/knockout verification: Compare staining patterns in wild-type versus JAM2 knockdown/knockout samples to confirm signal specificity.

• Western blot analysis: Verify the antibody detects bands at expected molecular weights (33 kDa and potentially 43 kDa due to glycosylation) . The JAM-B antibody should detect JAM2 protein consistently across different cell types, as demonstrated in the validation data showing detection in both Neuro2A and GL261 cell lysates .

• Peptide competition: Pre-incubate the antibody with the immunizing peptide/protein to confirm signal elimination when the antibody is neutralized.

• Cross-reactivity testing: If working with multiple species, confirm the antibody performs as expected across all target species. Many JAM2 antibodies have been validated for human, mouse, and rat samples .

What sample preparation methods optimize JAM2 detection?

Optimizing sample preparation for JAM2 detection depends on the experimental approach:

For Western blotting:

• Use lysis buffers containing protease inhibitors to prevent degradation

• Consider using RIPA or NP-40 based buffers which effectively solubilize membrane proteins

• Include phosphatase inhibitors if studying phosphorylation states

• Load approximately 30 μg of whole cell lysate, as demonstrated in successful validation studies

For immunohistochemistry:

• Both paraffin-embedded and frozen sections have been validated for JAM2 detection

• For paraffin sections, antigen retrieval methods may be necessary to expose epitopes

• Freshly prepared tissues generally yield better results than archived samples

For cell-based assays:

• Gentle fixation protocols are recommended to preserve membrane integrity

• Permeabilization should be optimized to access intracellular epitopes while maintaining structure

Why might I observe multiple bands when using JAM2 antibodies in Western blotting?

The detection of multiple bands with JAM2 antibodies is a common occurrence that can be attributed to several factors:

• Post-translational modifications: JAM2 undergoes glycosylation, which can result in detection at both 33 kDa (calculated molecular weight) and 43 kDa (glycosylated form) .

• Alternative splice variants: Up to three different isoforms of JAM2 have been reported, which may appear as distinct bands .

• Proteolytic processing: JAM2 may undergo partial degradation during sample preparation, resulting in smaller fragments.

• Cross-reactivity: Some antibodies might cross-react with related JAM family members (JAM1, JAM3) due to sequence homology.

To distinguish between these possibilities:

• Compare results across different cell/tissue types with known JAM2 expression patterns

• Use deglycosylation enzymes to confirm glycosylation-related shifts

• Implement gradient gels to better resolve closely migrating bands

• Consider using antibodies targeting different epitopes to confirm results

What strategies can resolve weak or inconsistent JAM2 signal in experiments?

Addressing weak or inconsistent JAM2 signal requires systematic optimization:

• Antibody concentration: Titrate antibody dilutions within the recommended range (1:500-1:2000 for Western blot) . Each testing system may require specific optimization to obtain optimal results.

• Incubation conditions: Adjust antibody incubation time and temperature. Overnight incubation at 4°C often improves signal quality compared to shorter incubations at room temperature.

• Detection system enhancement: For Western blots, consider using high-sensitivity ECL substrates. For IHC/ICC, evaluate signal amplification systems like tyramide signal amplification.

• Sample enrichment: For low-abundance targets, consider enriching the sample through immunoprecipitation before analysis or using tissue/cells with higher expression (heart, placenta) .

• Blocking optimization: Test different blocking agents (BSA, milk, commercial blockers) to reduce background while preserving specific signal.

• Antibody selection: If one antibody yields inconsistent results, evaluate alternative clones. For example, if a polyclonal antibody shows variability, switching to a well-characterized monoclonal (e.g., 7E4C5 or 1G4) might provide more consistent results.

How can JAM2 antibodies be utilized to investigate tight junction dynamics?

Investigating tight junction dynamics using JAM2 antibodies requires sophisticated approaches:

• Co-localization studies: Combine JAM2 antibodies with markers for other tight junction components (claudins, occludin, ZO-1) to examine spatial relationships and complex formation. This requires careful selection of compatible antibodies from different host species to allow simultaneous detection.

• Live-cell imaging: For dynamic studies, consider using non-perturbing JAM2 antibody fragments (Fab, scFv) conjugated to fluorophores for live imaging without disrupting junction integrity.

• Barrier function correlation: Pair JAM2 immunolocalization with functional assays of barrier integrity (TEER, dextran permeability) to correlate molecular organization with functional outcomes.

• Stimulus-induced reorganization: Track JAM2 redistribution following physiological stimuli or pathological challenges that affect endothelial barrier function, as JAM2 is critical for maintaining tight junction integrity between high endothelial cells .

• Super-resolution microscopy: Implement advanced imaging techniques (STED, STORM, PALM) to resolve nanoscale organization of JAM2 within junction complexes beyond the diffraction limit.

• Proximity ligation assays: Use this technique to visualize and quantify protein-protein interactions between JAM2 and potential binding partners at tight junctions with single-molecule sensitivity.

What approaches can reveal JAM2's role in lymphocyte homing mechanisms?

Investigating JAM2's involvement in lymphocyte homing requires integrative approaches:

• Adhesion assays: Use JAM2 antibodies to block specific epitopes and assess their impact on lymphocyte adhesion to endothelial cells under static and flow conditions.

• Transendothelial migration: Apply JAM2 antibodies in transmigration assays to determine their effect on lymphocyte passage across endothelial barriers, directly testing JAM2's role in this process.

• In vivo tracking: Administer fluorescently-labeled lymphocytes with/without JAM2 antibody pre-treatment and track their homing to secondary lymphoid organs using intravital microscopy.

• Receptor identification: Employ JAM2 antibodies in combination with candidate lymphocyte receptors to determine binding partners through co-immunoprecipitation, proximity ligation, or FRET-based approaches.

• Tissue-specific analysis: Compare JAM2 expression and function across different vascular beds, particularly high endothelial venules of lymph nodes where specialized endothelial cells facilitate lymphocyte extravasation .

• Conditional knockout models: Use tissue-specific JAM2 knockout models combined with antibody-based detection of remaining JAM2 to assess functional consequences on lymphocyte trafficking.

How can post-translational modifications of JAM2 be studied using antibodies?

Studying JAM2 post-translational modifications (PTMs) requires specialized antibody-based approaches:

• Modification-specific antibodies: Utilize antibodies specifically recognizing glycosylated JAM2 to distinguish between modified and unmodified forms. This can help explain the observed difference between calculated (33 kDa) and observed (43 kDa) molecular weights .

• Sequential immunoprecipitation: First immunoprecipitate total JAM2 using a pan-JAM2 antibody, then probe with modification-specific antibodies (anti-phospho, anti-glyco, etc.) to quantify modified fractions.

• Enzymatic treatment comparison: Compare JAM2 immunodetection patterns before and after treatment with deglycosylation enzymes (PNGase F, O-glycosidase) to map glycosylation sites and their functional importance.

• Mass spectrometry validation: Immunoprecipitate JAM2 using validated antibodies like those found in products with high validation counts , then analyze by mass spectrometry to identify specific modification sites.

• Site-directed mutagenesis correlation: Generate JAM2 constructs with mutations at predicted modification sites, express in model systems, then use JAM2 antibodies to assess how modifications affect localization, stability, and function.

How do JAM2 antibody detection patterns compare across different tissue types?

JAM2 expression varies significantly across tissues, with implications for experimental design and data interpretation:

• Tissue-specific expression levels: Highest expression is reported in heart, placenta, lung, foreskin, and lymph nodes . When designing experiments, consider these differences when selecting positive controls and interpreting signals across tissue panels.

• Cell-type specificity: JAM2 is predominantly expressed in vascular endothelial cells, particularly at tight junctions . In complex tissues, co-staining with endothelial markers can help distinguish true signal from background.

• Subcellular localization patterns: While primarily localized to tight junctions and cell membranes as a single-pass type I membrane protein , the distribution pattern may vary between polarized and non-polarized cells. This necessitates careful interpretation of immunostaining patterns.

• Detection sensitivity thresholds: In tissues with lower expression, signal amplification methods may be necessary to detect JAM2. Validation data from heart and placenta tissues provides useful reference points for expected signal intensity .

• Pathological alterations: Consider how disease states may alter JAM2 expression or localization, potentially affecting antibody detection patterns compared to normal tissue controls.

What considerations are important when selecting between monoclonal and polyclonal JAM2 antibodies?

The choice between monoclonal and polyclonal JAM2 antibodies significantly impacts experimental outcomes: