HMGB2 Antibody

What is HMGB2 and why is it significant in research contexts?

HMGB2 (High Mobility Group Box 2) is a highly conserved DNA-interacting protein that belongs to a family of proteins containing HMG box domains. While primarily nuclear, HMGB2 can also have extracellular actions during inflammation. Despite sharing high homology with HMGB1 (approximately 97% sequence similarity in mice), HMGB2 may have distinct cellular roles, particularly in neutrophils where it shows unique subcellular localization patterns compared to HMGB1 . Recent research has implicated HMGB2 in disease mechanisms such as IgA nephropathy, where it promotes APRIL expression by interacting with HMGA1 .

How can I validate HMGB2 antibody specificity when HMGB1 cross-reactivity is a concern?

Validating HMGB2 antibody specificity is crucial due to potential cross-reactivity with the highly homologous HMGB1 protein. A rigorous validation approach should include:

• Western blotting with both recombinant HMGB1 and HMGB2 proteins under reducing and non-reducing conditions

• Testing antibodies on knockout cell lines (HMGB1−/− and HMGB2−/− MEFs)

• Comparing multiple commercial antibodies side-by-side

Research shows that many commercial anti-HMGB1 antibodies readily detect recombinant HMGB2 to varying degrees. For example, antibodies such as MAB1690 and 6893 demonstrated significant cross-reactivity with HMGB2, while others showed weaker cross-reactivity . When selecting antibodies, prioritize those validated with knockout controls showing clear specificity for either protein.

What are the recommended HMGB2 antibodies with demonstrated specificity in research applications?

Based on validation studies and citation records, the following HMGB2 antibodies have demonstrated reliability:

Among these, Proteintech's 14597-1-AP has the most extensive validation data and citations, with demonstrated reactivity in human, mouse, and rat samples across multiple applications .

What are the optimal dilutions and experimental conditions for different HMGB2 antibody applications?

Proper dilution and experimental conditions are critical for successful HMGB2 detection. Based on validation studies, the following recommendations apply for the widely-used Proteintech 14597-1-AP antibody:

Always titrate the antibody in your specific experimental system to obtain optimal results, as performance may vary between tissue and cell types.

How should I design controls when studying HMGB2 expression in primary cells versus cell lines?

Designing appropriate controls is crucial for accurate HMGB2 expression analysis:

• For cell lines:

  • Positive control: Use cell lines with confirmed HMGB2 expression (HEK-293, HepG2, Jurkat)

  • Negative control: If possible, use HMGB2 knockout cells (HMGB2−/− MEFs)

  • Loading control: Standard housekeeping proteins (β-actin, GAPDH)

  • Specificity control: Recombinant HMGB2 protein competition assay

• For primary cells:

  • Compare expression across multiple donors

  • Include relevant IgG controls at equivalent concentrations

  • Consider cell-type specific expression patterns (e.g., neutrophils show distinct nuclear HMGB2 localization)

  • Validate with multiple techniques (WB, IF, qRT-PCR)

Research has demonstrated that HMGB2 can have different subcellular locations in different cell types. For instance, while both HMGB1 and HMGB2 are nuclear in endothelial cells, HMGB2 remains nuclear in neutrophils while HMGB1 is predominantly cytoplasmic , highlighting the importance of cell-type specific controls.

What are common challenges in detecting HMGB2 and how can they be resolved?

Researchers frequently encounter these challenges when working with HMGB2 antibodies:

• Molecular weight discrepancy:

  • Problem: HMGB2 has a calculated MW of 24 kDa but is typically observed at 33-35 kDa on Western blots

  • Solution: This is normal due to post-translational modifications and protein structure; confirm with positive controls

• Cross-reactivity with HMGB1:

  • Problem: Many antibodies detect both proteins due to high sequence homology

  • Solution: Use antibodies validated with knockout cells; consider parallel detection with specific HMGB1 and HMGB2 antibodies

• Redox sensitivity:

  • Problem: Some antibodies show differential affinity for reduced versus non-reduced forms

  • Solution: Maintain consistent sample preparation conditions; Ab92310 particularly shows preference for reduced HMGB1

• Nuclear versus cytoplasmic staining:

  • Problem: Variable localization patterns across cell types

  • Solution: Include appropriate subcellular markers; perform fractionation experiments to confirm localization

How can I optimize immunofluorescence protocols to distinguish between HMGB1 and HMGB2 in co-expression studies?

When studying both HMGB proteins simultaneously, consider these optimization strategies:

• Antibody selection:

  • Use antibodies raised in different host species (e.g., rabbit anti-HMGB2, mouse anti-HMGB1)

  • Validate specificity with blocking peptides or knockout controls

  • Select antibodies targeting non-homologous regions

• Staining protocol:

  • Sequential rather than simultaneous staining may reduce cross-reactivity

  • Include specific blocking steps with excess non-labeled antibody

  • Optimize fixation methods (PFA versus methanol can affect epitope accessibility)

• Imaging optimization:

  • Use confocal microscopy with appropriate controls for bleed-through

  • Perform single-stain controls to verify specificity

  • Quantitative colocalization analysis with appropriate statistical metrics

Research has shown that in neutrophils, HMGB1 is cytoplasmic while HMGB2 remains nuclear, providing a useful model system to validate co-staining protocols. In contrast, both proteins are nuclear in endothelial cells, requiring more stringent specificity controls .

How can I design experiments to investigate HMGB2's role in transcriptional regulation?

HMGB2 functions as an architectural transcription factor. To study its regulatory role:

• Chromatin immunoprecipitation (ChIP) approaches:

  • Use validated HMGB2 antibodies (e.g., 14597-1-AP) for ChIP-seq

  • Include appropriate IgG controls

  • Consider dual ChIP to identify co-binding with interacting partners (e.g., HMGA1)

• Functional validation approaches:

  • RNA interference (knockdown) of HMGB2 followed by transcriptome analysis

  • DNA pull-down assays with predicted binding regions

  • Coimmunoprecipitation (Co-IP) to identify protein-protein interactions

• Target gene analysis:

  • Focus on genes with known HMGB2 binding sites

  • Validate with reporter assays

  • Assess binding site mutations to confirm specificity

Recent research demonstrated that HMGB2 regulates APRIL expression by interacting with HMGA1, which subsequently leads to increased Gd-IgA1 expression in IgA nephropathy . Similar experimental approaches could be applied to other disease contexts.

What are the current methods for studying HMGB2's role in inflammation and immune responses?

HMGB2 has emerging roles in inflammation. To investigate these functions:

• Extracellular versus intracellular functions:

  • Distinguish between nuclear and extracellular HMGB2 using subcellular fractionation

  • Measure secreted HMGB2 in conditioned media using sensitive ELISAs

  • Compare redox forms using non-reducing versus reducing conditions

• Cell-specific expression and function:

  • Analyze immune cell subtypes (neutrophils show distinct HMGB2 localization)

  • Use flow cytometry with specific antibodies to quantify expression levels

  • Consider single-cell approaches to capture heterogeneity

• In vivo models:

  • Use conditional knockout models to assess tissue-specific functions

  • Apply neutralizing antibodies to block extracellular functions

  • Correlate findings with human inflammatory conditions

The recent finding that HMGB2 expression is elevated in peripheral blood mononuclear cells from IgA nephropathy patients and correlates with disease severity suggests its potential role as a biomarker or therapeutic target in inflammatory diseases .

How can I investigate the differential roles of HMGB1 versus HMGB2 in disease pathogenesis?

Despite high sequence homology, HMGB1 and HMGB2 may have distinct functions in disease contexts:

• Comparative expression analysis:

  • Quantify relative expression ratios in healthy versus diseased tissues

  • Analyze cell-type specific expression patterns

  • Assess subcellular localization differences across disease stages

• Selective targeting approaches:

  • Use specific antibodies validated against knockout controls

  • Design selective inhibitors targeting non-homologous regions

  • Apply isoform-specific RNA interference

• Interaction network analysis:

  • Identify unique binding partners through IP-mass spectrometry

  • Compare chromatin binding profiles via ChIP-seq

  • Analyze post-translational modifications that differ between isoforms

Research has shown that in IgA nephropathy, HMGB2 promotes APRIL expression by interacting with HMGA1, inducing Gd-IgA1 overexpression and contributing to disease pathogenesis , suggesting a specific role distinct from HMGB1 in this context.

What methodological approaches are recommended for studying HMGB2's interaction with binding partners like HMGA1?

To investigate protein-protein interactions involving HMGB2:

• Co-immunoprecipitation approaches:

  • Use specific antibodies against HMGB2 (e.g., Proteintech 14597-1-AP)

  • Confirm specificity with knockout controls or competitive blocking

  • Perform reciprocal IP with antibodies against suspected partners (e.g., HMGA1)

• Proximity-based methods:

  • Consider proximity ligation assays for in situ detection

  • FRET/BRET approaches with tagged proteins

  • BioID or APEX2 proximity labeling to identify interaction networks

• Functional validation:

  • Knockdown studies targeting HMGB2, partners, or both

  • Structure-function analysis with domain mutants

  • Competitive peptide inhibition of specific interaction domains

Recent research used coimmunoprecipitation to demonstrate that HMGB2 binds to HMGA1, which then binds to the promoter region of the APRIL gene. RNA interference experiments showed that HMGA1 knockdown reduced Gd-IgA1 concentration in cell supernatants, suggesting a functional consequence of this interaction .

What are the current limitations in HMGB2 antibody research and how might future technologies address them?

Current challenges in HMGB2 antibody research include:

• Cross-reactivity issues:

  • Most commercial antibodies show some degree of HMGB1 cross-reactivity

  • Limited epitope mapping information from manufacturers

  • Varying specificity under different experimental conditions

• Structural and functional heterogeneity:

  • Different redox forms may have distinct functions

  • Post-translational modifications affect antibody recognition

  • Conformational changes upon binding partners

Future technological advances may include:

• Development of highly specific monoclonal antibodies targeting unique epitopes

• Recombinant antibody fragments with enhanced specificity

• Structure-based design of isoform-selective nanobodies

• Advanced proteomics approaches to characterize HMGB2 complexes

How should researchers design experiments to investigate HMGB2's role in emerging fields like cancer immunology or neuroinflammation?

As HMGB2 research expands into new areas, consider these experimental approaches:

• In cancer research:

  • Analyze HMGB2 expression across cancer types and stages

  • Investigate correlation with immune infiltration patterns

  • Assess impact on treatment response through knockdown studies

  • Explore potential as a biomarker or therapeutic target

• In neuroinflammation:

  • Compare HMGB2 versus HMGB1 expression in neurological disorders

  • Analyze cell-type specific functions (neurons, glia, infiltrating immune cells)

  • Investigate blood-brain barrier crossing capabilities

  • Study the impact of HMGB2 neutralization in disease models

• Translational approaches:

  • Develop specific bioassays to quantify HMGB2 in clinical samples

  • Design therapeutic strategies targeting specific HMGB2 functions

  • Explore combinatorial approaches targeting multiple DAMP molecules