HMGN2 Antibody

What is HMGN2 and what are its primary biological functions?

HMGN2 is a highly conserved nucleosomal protein that plays a critical role in unfolding higher-order chromatin structure and facilitating transcriptional activation of mammalian genes. It is one of the most abundant, ubiquitous, and evolutionarily conserved non-histone proteins found in the nucleus . HMGN2 binds nucleosomal DNA and is associated with transcriptionally active chromatin regions .

The protein exhibits a calculated molecular weight of 9 kDa (or 9aa, 3 kDa according to some sources), though its observed molecular weight in experimental settings typically ranges from 17-20 kDa . This difference likely reflects post-translational modifications or the protein's biophysical properties.

Beyond its nuclear function in chromatin regulation, HMGN2 has been identified as an anti-tumor effector molecule released by CD8+ T cells. Research has demonstrated that HMGN2 can induce tumor cell apoptosis at low doses and inhibit the growth of various cancer cell lines including Tca8113, adenoid cystic carcinoma cell-2 line (ACC-2), human lung adenocarcinoma epithelial cell line A549, and bladder cancer cell line T24 .

How do HMGN2 antibodies differ in terms of host species, clonality, and applications?

HMGN2 antibodies are primarily available as polyclonal antibodies raised in rabbits, though the specific characteristics can vary based on the manufacturer and production methods. The antibodies typically demonstrate reactivity against human HMGN2, with some showing cross-reactivity with mouse and pig samples .

In terms of applications, HMGN2 antibodies have been validated for multiple experimental techniques:

The antibodies are typically purified using antigen affinity methods and supplied in liquid form with specific storage buffers (e.g., PBS with 0.02% sodium azide and 50% glycerol pH 7.3) . This formulation helps maintain antibody stability and functionality during storage and experimental use.

What control samples should be included when working with HMGN2 antibodies?

When designing experiments with HMGN2 antibodies, appropriate controls are essential for result validation. Based on the literature, several cell lines have been demonstrated to express detectable levels of HMGN2 and can serve as positive controls:

• Jurkat cells

• HeLa cells

• HepG2 cells

• HL-60 cells

• K-562 cells

• 293T cells

• MCF7 cells

For tissue-based applications, human prostate cancer tissue and human cervical cancer tissue have shown positive staining with HMGN2 antibodies . When conducting T-cell activation studies, PHA-stimulated PBMCs provide a reliable positive control for HMGN2 expression, particularly in the CD8+ T cell population .

Negative controls should include isotype controls (rabbit IgG at equivalent concentrations) and, when possible, samples with HMGN2 knockdown or knockout. Additionally, blocking experiments using recombinant HMGN2 protein can verify antibody specificity.

What is the recommended protocol for detecting HMGN2 by Western blot?

For optimal Western blot detection of HMGN2, the following methodology is recommended:

• Sample preparation: Lyse cells in a buffer containing protease inhibitors. HMGN2 is primarily localized in the nucleus and cytoplasm, so ensure complete cell lysis.

• Gel electrophoresis: Use 12-15% SDS-PAGE gels due to the relatively small size of HMGN2 (observed molecular weight: 17-20 kDa).

• Transfer and blocking: Transfer proteins to a PVDF or nitrocellulose membrane using standard protocols. Block with 5% non-fat milk or BSA in TBST.

• Primary antibody incubation: Dilute HMGN2 antibody at 1:500-1:1000 (or up to 1:2000 depending on the specific antibody) . Incubate overnight at 4°C or for 1-2 hours at room temperature.

• Secondary antibody and detection: Use an appropriate HRP-conjugated secondary antibody (anti-rabbit IgG) and detect using chemiluminescence.

Note that HMGN2 typically appears as a band at 17-20 kDa, which is higher than its calculated molecular weight of 9 kDa. This discrepancy is common for many nuclear proteins and may be due to post-translational modifications or the protein's biophysical properties .

How should intracellular staining for HMGN2 be performed for flow cytometry analysis?

For intracellular staining of HMGN2 in cells for flow cytometry analysis, the following protocol has been validated:

• Surface marker staining: If analyzing specific cell populations, first stain cells with fluorescence-labeled surface markers (e.g., CD4-PE, CD8-PE, or CD44-APC for T cell subsets) .

• Fixation: Add 100 μl fixation buffer to fix cells and incubate at 4°C overnight.

• Permeabilization: Wash cells three times with permeabilization buffer to ensure access to intracellular antigens.

• Primary antibody: Add rabbit anti-human HMGN2 antibody (1 μg/ml) to permeabilized cells. Include a parallel sample with PBS instead of primary antibody as a negative control.

• Secondary antibody: After washing, add fluorochrome-conjugated anti-rabbit IgG secondary antibody (e.g., goat anti-rabbit IgG-FITC).

• Analysis: Analyze samples by flow cytometry, gating on relevant cell populations if surface markers were used.

When analyzing activated T cells, using CD44high as an activation marker can help identify the subpopulation of interest. Research has shown that after PHA stimulation, HMGN2 expression increases significantly in both CD4+ and CD8+ T cells, with CD8+ T cells showing higher expression (50.71 ± 10.34%) compared to CD4+ T cells (16.67 ± 5.61%) .

What protocol is recommended for quantifying HMGN2 by ELISA?

For quantitative analysis of HMGN2 in supernatants or cellular extracts, the following ELISA protocol has been validated:

• Plate coating: Coat ELISA plates with 100 μl of samples, HMGN2 protein standards (prepared at different concentrations), or PBS (negative control). Incubate overnight at 4°C.

• Washing and blocking: Wash plates three times with wash buffer. Block with appropriate blocking buffer (typically 1-5% BSA in PBS).

• Primary antibody: Add 100 μl of rabbit anti-human HMGN2 antibody (1:500 dilution) and incubate at 37°C for 1 hour.

• Secondary antibody: After thorough washing, add 100 μl of HRP-conjugated anti-rabbit IgG secondary antibody (1:1000 dilution) and incubate at 37°C for 1 hour.

• Detection: Add 100 μl TMB substrate solution to each well. After 20 min incubation, stop the reaction with 2N H2SO4 and measure absorbance at 490 nm using an ELISA plate reader .

This method has been successfully used to quantify HMGN2 released by activated T cells, with concentrations ranging from approximately 300-550 ng/ml in different cell populations .

How is HMGN2 involved in anti-tumor immune responses?

HMGN2 has emerged as an important anti-tumor effector molecule of CD8+ T cells. Research has established several key mechanisms of its involvement in anti-tumor immunity:

• Release by activated T cells: Both PHA-stimulated and tumor antigen-activated CD8+ T cells release high levels of HMGN2. CD8+ T cells are the major cell population in PBMCs that release HMGN2 after activation .

• Direct tumor cytotoxicity: Supernatants of tumor antigen-activated CD8+ T cells containing HMGN2 can kill tumor cells in a dose-dependent manner. This anti-tumor effect can be significantly blocked using an anti-HMGN2 antibody, confirming HMGN2's direct role in tumor cell killing .

• Cellular uptake mechanism: Fluorescence-labeling assays have demonstrated that HMGN2 from activated CD8+ T cells can be transported into tumor cells. This transport is visibly decreased after HMGN2 is depleted by anti-HMGN2 antibody, suggesting a specific uptake mechanism .

• Apoptosis induction: HMGN2 has been shown to inhibit the growth of multiple cancer cell lines, including Tca8113, ACC-2, A549, and T24, by promoting apoptosis both in vitro and in vivo .

• Tumor vasculature targeting: A 31-residue peptide fragment of HMGN2 (called F3, corresponding to amino acids 17-48) has been identified as a potent homing peptide that selectively binds to tumor cells both in vitro and in vivo, suggesting a role in targeting tumor vasculature .

These findings collectively establish HMGN2 as a novel anti-tumor effector molecule with potential implications for cancer immunotherapy.

What methods can be used to evaluate the anti-tumor effects of HMGN2?

Several validated experimental approaches can be employed to assess the anti-tumor effects of HMGN2:

• Tumor cell cytotoxicity assays:

  • Treat tumor cell lines with purified HMGN2 or supernatants from activated CD8+ T cells

  • Measure cell viability using MTT/XTT assays, flow cytometry with annexin V/PI staining, or other cytotoxicity readouts

  • Include control conditions with HMGN2-depleted supernatants (using anti-HMGN2 antibodies) to confirm specificity

• Cellular uptake studies:

  • Label purified HMGN2 with fluorescent markers

  • Treat tumor cells and observe uptake using confocal microscopy

  • Compare uptake in the presence and absence of blocking antibodies

• Apoptosis mechanism analysis:

  • Assess key apoptotic markers (caspase activation, PARP cleavage, etc.) in HMGN2-treated tumor cells

  • Evaluate changes in mitochondrial membrane potential

  • Analyze DNA fragmentation as a hallmark of apoptosis

• In vivo tumor models:

  • Establish xenograft or syngeneic tumor models

  • Administer purified HMGN2 or adoptively transfer HMGN2-producing CD8+ T cells

  • Monitor tumor growth, survival, and infiltrating immune cells

  • Include controls with HMGN2-neutralizing antibodies

• Tumor vasculature targeting:

  • Evaluate the binding of HMGN2 or its F3 peptide fragment to tumor endothelial cells

  • Assess vascular targeting in tumor tissue sections after systemic administration of labeled HMGN2

These methodologies provide a comprehensive framework for investigating the anti-tumor properties of HMGN2 and its potential as a therapeutic target.

How can researchers isolate and analyze HMGN2 from activated T cells?

To isolate and analyze HMGN2 from activated T cells, researchers can follow these validated approaches:

• T cell activation and isolation:

  • Isolate PBMCs from healthy donors using density gradient centrifugation

  • Activate T cells using either:

    • PHA (20 μg/ml) + IL-2 (100 IU/ml) for 72 hours, or

    • Tumor antigens (150 μg/ml tumor full protein) for 7 days

  • For specific T cell subsets, use:

    • Surface marker staining (CD3, CD8, CD4) followed by FACS sorting

    • Magnetic bead-based isolation systems

• HMGN2 detection and quantification:

  • Collect supernatants for secreted HMGN2

  • Prepare cell lysates for intracellular HMGN2

  • Analyze HMGN2 levels using:

    • ELISA (see protocol in section 2.3)

    • Western blot with anti-HMGN2 antibody

    • Intracellular staining for flow cytometry (see protocol in section 2.2)

• Functional analysis of isolated HMGN2:

  • Assess anti-tumor activity of T cell supernatants

  • Perform blocking experiments with anti-HMGN2 antibodies

  • Compare activity between different T cell subsets

In published protocols, CD8+ T cells activated with PHA demonstrated the highest HMGN2 expression (68.37 ± 15.21% by intracellular staining; 539.00 ± 118 ng/ml in supernatants) compared to CD4+ T cells and other PBMC populations . Tumor antigen-activated CD8+ T cells also showed high levels of HMGN2, particularly in the CD44high activated subpopulation .

How can researchers investigate the role of HMGN2 in chromatin remodeling and gene regulation?

To investigate HMGN2's role in chromatin remodeling and gene regulation, researchers can employ these advanced approaches:

• Chromatin Immunoprecipitation (ChIP) assays:

  • Use validated HMGN2 antibodies to immunoprecipitate HMGN2-bound chromatin

  • Perform ChIP-seq to identify genome-wide binding sites

  • Compare HMGN2 binding patterns with active chromatin marks (H3K4me3, H3K27ac)

  • Correlate binding sites with transcriptionally active regions

• Gene expression analysis after HMGN2 modulation:

  • Perform HMGN2 knockdown using siRNA/shRNA or CRISPR-Cas9

  • Overexpress HMGN2 using suitable expression vectors

  • Analyze changes in gene expression using RNA-seq or targeted qPCR

  • Compare effects on genes involved in different cellular processes (proliferation, differentiation, immune response)

• Nucleosome positioning and accessibility studies:

  • Use ATAC-seq or MNase-seq to assess chromatin accessibility

  • Compare chromatin structure in the presence and absence of HMGN2

  • Analyze nucleosome positioning around HMGN2 binding sites

• Protein-protein interaction analysis:

  • Perform co-immunoprecipitation with HMGN2 antibodies

  • Identify interaction partners using mass spectrometry

  • Validate interactions with key chromatin modifiers or transcription factors

  • Map interaction domains through deletion mutants

• Live-cell imaging of chromatin dynamics:

  • Generate fluorescently tagged HMGN2 constructs

  • Visualize HMGN2 mobility and chromatin association in living cells

  • Assess changes in response to cellular stimuli or during cell cycle progression

These methodologies can provide comprehensive insights into how HMGN2 influences chromatin structure and gene expression in normal and disease states .

What factors should be considered when contradictory results emerge from different HMGN2 detection methods?

When researchers encounter contradictory results between different HMGN2 detection methods, several factors should be considered:

• Antibody specificity and epitope accessibility:

  • Different antibodies may recognize distinct epitopes that could be differentially accessible in various experimental conditions

  • Perform validation using multiple antibodies targeting different regions of HMGN2

  • Include appropriate positive and negative controls in all experiments

• Post-translational modifications:

  • HMGN2 may undergo various post-translational modifications affecting antibody recognition

  • The significant difference between calculated (9 kDa) and observed (17-20 kDa) molecular weight suggests modifications or specific biophysical properties

  • Consider using modification-specific antibodies if available

• Cell type-specific expression and localization:

  • HMGN2 expression and subcellular localization may vary across cell types

  • CD8+ T cells show higher HMGN2 expression than CD4+ T cells after activation

  • Ensure appropriate cell fractionation when analyzing nuclear vs. cytoplasmic pools

• Activation state-dependent expression:

  • HMGN2 levels change significantly upon cellular activation

  • Only CD44high (activated) CD8+ T cells show high HMGN2 expression after tumor antigen stimulation

  • Standardize activation protocols and timing for consistent results

• Technical considerations for specific methods:

  • For Western blot: Sample preparation, gel percentage, transfer conditions

  • For flow cytometry: Fixation/permeabilization protocols, compensation settings

  • For ELISA: Coating conditions, antibody concentrations, detection systems

  • For IHC/IF: Antigen retrieval methods (TE buffer pH 9.0 recommended for HMGN2)

When discrepancies arise, employing orthogonal detection methods and standardizing experimental conditions across laboratories can help resolve contradictions and establish consensus findings.

What are the key considerations for studying HMGN2 in disease models and potential therapeutic applications?

When investigating HMGN2 in disease models and potential therapeutic applications, researchers should consider these critical factors:

• Selection of appropriate disease models:

  • For cancer studies: Choose models that reflect the tumor types where HMGN2 has shown efficacy (e.g., Tca8113, ACC-2, A549, T24 cell lines)

  • Consider both in vitro cell lines and in vivo xenograft or syngeneic models

  • For immunotherapy applications: Use immunocompetent models to study HMGN2's interaction with the host immune system

• Delivery strategies for HMGN2-based therapeutics:

  • Leverage the tumor-homing properties of the F3 peptide (amino acids 17-48 of HMGN2)

  • Evaluate direct delivery of recombinant HMGN2 versus adoptive transfer of HMGN2-producing T cells

  • Assess stability, pharmacokinetics, and tissue distribution of HMGN2-based agents

• Combination therapy approaches:

  • Test HMGN2 in combination with established cancer therapies (chemotherapy, radiation, immunotherapy)

  • Investigate potential synergies with other immune-activating strategies

  • Evaluate combined targeting of multiple tumor pathways

• Monitoring immune responses:

  • Track changes in tumor-infiltrating lymphocytes after HMGN2 treatment

  • Assess broader immune activation or potential adverse immune effects

  • Evaluate memory responses and long-term anti-tumor immunity

• Translational considerations:

  • Develop robust biomarkers for patient selection and response monitoring

  • Establish humanized models to better predict clinical responses

  • Address manufacturing and stability challenges for clinical development

  • Evaluate potential immunogenicity of HMGN2-based therapeutics

By systematically addressing these considerations, researchers can advance the understanding of HMGN2's role in disease pathogenesis and explore its potential as a therapeutic agent, particularly in cancer immunotherapy contexts where its anti-tumor effects have been demonstrated .