HMGB1 Recombinant Monoclonal Antibody

What is HMGB1 and why is it an important research target?

HMGB1 is a 25-30 kDa non-histone chromosomal protein that functions both as a nuclear DNA-binding protein and as an extracellular damage-associated molecular pattern (DAMP). In the nucleus, HMGB1 bends DNA and regulates gene expression by stabilizing nucleosome formation and recruiting transcription factors . Extracellularly, it acts as an inflammatory mediator that promotes monocyte migration and cytokine secretion, as well as facilitates T cell-dendritic cell interactions .

HMGB1 has emerged as an important research target because:

• It is expressed at high levels in almost all cells

• It translocates from the nucleus to the cytoplasm during autophagy

• It can be passively released from necrotic cells or actively secreted from various immune and non-immune cells

• It signals through receptors including RAGE, TLR2, and TLR4, triggering inflammatory responses

• It has been implicated in numerous pathological conditions, particularly autoimmune diseases like multiple sclerosis

Selecting the appropriate HMGB1 antibody requires consideration of several factors:

• Species reactivity: Verify that the antibody recognizes HMGB1 in your species of interest. Many HMGB1 antibodies cross-react with human, mouse, and rat HMGB1 due to high sequence homology .

• Application compatibility: Ensure the antibody is validated for your specific application. Not all antibodies work equally well across different applications .

• Epitope specificity: Consider which domain of HMGB1 you need to target:

  • Antibodies targeting the A-box may be useful for studying transcription factor interactions

  • Antibodies targeting the B-box can block cytokine activity

  • C-terminal antibodies may detect different post-translational modifications

• Validated samples: Review antibody datasheets for verified positive controls. For example, the E-AB-81436 antibody has been verified in K562, rat brain, C6, 3T3, and HeLa cell lysates .

• Clone selection: Different monoclonal clones (e.g., Giby-1-4, 951420, DPH1.1) may have different properties and specificities .

What are the optimal dilution ranges for HMGB1 antibodies in different applications?

Optimal dilution ranges vary by application and specific antibody:

Always perform a dilution series to determine the optimal concentration for your specific experimental conditions and samples.

How does HMGB1 cellular localization affect antibody selection and experimental design?

HMGB1 exhibits complex cellular localization patterns that must be considered:

• Nuclear localization: In basal states, HMGB1 is predominantly nuclear

• Cytoplasmic translocation: During cell activation or stress, HMGB1 shuttles to the cytoplasm

• Secretion: HMGB1 can be actively secreted via secretory endolysosome exocytosis

• Extracellular release: HMGB1 is passively released from necrotic cells or actively from immune cells

This dynamic localization affects experimental design:

• For nuclear HMGB1 studies, nuclear extraction protocols are essential

• For extracellular HMGB1, culture supernatants or serum/plasma samples should be collected

• Fixation methods for microscopy can affect HMGB1 localization visualization

• Time course experiments may be necessary to capture translocation events

How can HMGB1 monoclonal antibodies be used in autoimmune disease research?

HMGB1 monoclonal antibodies have proven valuable in autoimmune disease research, particularly in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis:

• Therapeutic intervention studies: Anti-HMGB1 monoclonal antibodies (20 μg administered intraperitoneally for 5 consecutive days) significantly ameliorated clinical severity and pathological features of EAE in mouse models .

• Mechanism investigations: Treatment with anti-HMGB1 antibodies:

  • Reduced infiltration of inflammatory cells into the central nervous system

  • Decreased demyelination in spinal cord tissue

  • Attenuated IL-17 upregulation in serum

  • Preserved nuclear HMGB1 immunoreactivity in spinal cord cells

• Translational potential: The successful treatment of EAE with anti-HMGB1 antibodies suggests that targeting HMGB1 could represent a novel therapeutic strategy for multiple sclerosis in humans .

• Experimental design considerations:

  • Control groups should include isotype-matched IgG administration (e.g., 20 μg mouse IgG)

  • Timing of antibody administration is critical (e.g., days 11-15 after EAE induction)

  • Multiple parameters should be evaluated: clinical scoring, histopathology, and cytokine profiling

What technical considerations are important when detecting both nuclear and extracellular HMGB1?

Detecting both nuclear and extracellular HMGB1 presents unique technical challenges:

• Sample preparation:

  • For nuclear HMGB1: Nuclear extraction protocols must preserve nuclear integrity

  • For extracellular HMGB1: Cell culture supernatants, serum, or tissue fluids must be collected with protease inhibitors to prevent degradation

• Immunohistochemistry observations:

  • Normal tissue typically shows strong nuclear HMGB1 immunoreactivity

  • In disease states (e.g., EAE), nuclear HMGB1 immunoreactivity may be lost as HMGB1 translocates to the cytoplasm and extracellular space

  • Treatment with anti-HMGB1 antibodies can partially restore nuclear localization

• Redox state considerations:

  • HMGB1 is released from necrotic cells in a fully reduced state and subsequently becomes oxidized

  • Different redox states may affect antibody recognition

  • Some antibodies may preferentially detect specific redox forms

• Post-translational modifications:

  • Acetylation of HMGB1 (on up to 17 lysine residues) affects its localization and function

  • Secreted HMGB1 is typically in an acetylated form

  • Antibodies may differ in their ability to detect modified forms

How can I validate the specificity of my HMGB1 antibody?

Proper validation of HMGB1 antibody specificity is critical:

• Positive control samples: Use verified positive samples based on antibody datasheets:

  • Human cell lines: Jurkat, K562, HEK293, HeLa

  • Mouse cell lines: Hepa 1-6, 3T3

  • Rat samples: Rat brain, C6, H4-II-E-C3

• Western blot validation:

  • Confirm the expected molecular weight (approximately 25-28 kDa)

  • Reduced conditions are typically used for HMGB1 detection

  • Multiple bands may indicate different post-translational modifications

• Cross-reactivity testing:

  • Test against related proteins (e.g., HMGB2)

  • Some antibodies like DPH1.1 specifically recognize HMGB1 but not HMGB2

• Knockout/knockdown controls:

  • When available, use HMGB1 knockout or knockdown samples as negative controls

  • KO-validated antibodies provide higher confidence in specificity

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide

  • For example, DPH1.1 mAb was generated against the 17-mer peptide P1 (KGKPDAAKKGVVKAEKS)

  • This should abolish specific binding if the antibody is truly specific

What are the considerations for using HMGB1 antibodies in in vivo experiments?

When using HMGB1 antibodies for in vivo studies:

• Dosage and administration:

  • EAE studies used 20 μg of anti-HMGB1 monoclonal antibody intraperitoneally for 5 consecutive days

  • DPH1.1 mAb has been administered intravenously at 220 μg/mouse to block inflammatory cell recruitment

  • Timing of administration is critical and should be optimized for your specific model

• Controls:

  • Include isotype-matched IgG controls at equivalent doses

  • Vehicle control groups (e.g., PBS alone) should also be included

• Endpoint analyses:

  • Clinical scoring systems for disease models

  • Histopathological examination

  • Cytokine profiling (e.g., IL-4, IL-6, IL-10, IL-17, IFN-γ, TNF-α)

  • Cell trafficking and recruitment assays

• Species considerations:

  • Ensure the antibody recognizes HMGB1 in your animal model species

  • Most antibodies recognize human, mouse, and rat HMGB1 due to high sequence conservation

• Regulatory requirements:

  • Remember that antibodies for in vivo studies are intended for research only and cannot be used on humans

  • Follow institutional guidelines for animal experimentation

How do post-translational modifications of HMGB1 affect antibody recognition?

Post-translational modifications significantly impact HMGB1 function and antibody recognition:

• Acetylation:

  • HMGB1 can be acetylated on up to 17 lysine residues

  • Acetylation promotes nuclear export and secretion

  • Antibodies raised against unmodified peptides may have reduced affinity for acetylated HMGB1

• Redox state:

  • HMGB1 contains three cysteine residues (C23, C45, and C106) that can exist in different redox states

  • Fully reduced HMGB1 exhibits chemotactic activity

  • Disulfide HMGB1 (C23-C45) has cytokine-inducing activity

  • Fully oxidized HMGB1 has neither activity

  • Different antibodies may preferentially recognize specific redox forms

• Phosphorylation:

  • Phosphorylation of HMGB1 can affect its nuclear-cytoplasmic shuttling

  • Phospho-specific antibodies may be required for certain studies

• Technical implications:

  • Western blot may reveal multiple bands representing differently modified forms

  • Sample preparation should preserve the modification state of interest

  • Reducing or non-reducing conditions should be chosen based on the modification being studied

Why might I observe multiple bands when using HMGB1 antibodies in Western blotting?

Multiple bands in HMGB1 Western blots can occur for several reasons:

• Post-translational modifications: HMGB1 undergoes various modifications including acetylation (up to 17 sites), phosphorylation, and redox changes. Different modified forms can appear as separate bands .

• Mobility variations: The observed molecular weight of HMGB1 may vary from the calculated 25 kDa. This discrepancy is explained in search result : "The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size."

• Sample preparation: Different extraction methods (e.g., whole cell lysate vs. nuclear extract) can affect the HMGB1 forms detected.

• Proteolytic degradation: Improper sample handling or insufficient protease inhibitors can result in degradation products.

• Antibody cross-reactivity: Some antibodies may cross-react with related proteins like HMGB2, though specific antibodies like DPH1.1 do not recognize HMGB2 .

To address multiple bands:

• Use positive control samples with known HMGB1 expression

• Compare reducing and non-reducing conditions

• Consider using different lysis buffers to preserve specific modifications

• Test multiple antibodies targeting different epitopes

How can I optimize immunohistochemistry protocols for HMGB1 detection?

Optimizing IHC for HMGB1 requires attention to several details:

• Fixation and processing:

  • Paraffin embedding is commonly used for HMGB1 IHC

  • Immersion fixation in paraformaldehyde preserves HMGB1 localization

• Antibody selection and dilution:

  • IHC-P typically requires 1:50-1:100 dilution

  • 5 μg/mL concentration has been effective for human prostate cancer tissue

• Antigen retrieval:

  • Critical for detecting nuclear HMGB1

  • Heat-induced epitope retrieval methods are commonly used

• Detection systems:

  • HRP-DAB systems work well for HMGB1 visualization

  • Counterstaining with hematoxylin helps visualize nuclear vs. cytoplasmic localization

• Controls and interpretation:

  • Normal tissue typically shows strong nuclear HMGB1 immunoreactivity

  • In inflammatory or disease states, both nuclear and cytoplasmic/extracellular staining may be observed

  • HMGB1 staining has been validated in human prostate cancer tissue, human tonsil, and other tissues

• Step-by-step protocol example:

  • Follow the protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections as referenced in search result

  • Incubate with primary antibody overnight at 4°C for optimal results

What are the best methods for detecting extracellular HMGB1 in biological samples?

Detecting extracellular HMGB1 requires specific methodologies:

• Sample collection:

  • Cell culture supernatants: Collect after appropriate stimulation time

  • Serum/plasma: Process quickly and store with protease inhibitors

  • Cerebrospinal fluid: Particularly relevant for CNS studies like EAE

• ELISA:

  • Most direct method for quantifying soluble HMGB1

  • Commercial ELISA kits are available

  • Standard curves should be prepared using recombinant HMGB1

• Western blotting of concentrated supernatants:

  • TCA precipitation or other concentration methods may be needed

  • Typically requires 1:1000-1:2000 antibody dilution

• Multiplexed assays:

  • Fluorescent magnetic bead-based immunoassays allow simultaneous detection of HMGB1 and cytokines

  • Useful for correlating HMGB1 release with other inflammatory mediators

• In vivo release visualization:

  • Tissue sections can be examined for extranuclear HMGB1 staining

  • Loss of nuclear HMGB1 immunoreactivity in EAE correlates with release

• Consideration of HMGB1 redox state:

  • HMGB1 is released from necrotic cells in fully reduced form and subsequently oxidized

  • Different redox forms have different biological activities

What controls should be included when using HMGB1 antibodies?

Proper controls are essential for reliable HMGB1 studies:

• Positive controls:

  • Cell lines: Jurkat, K562, HEK293, HeLa (human); Hepa 1-6, 3T3 (mouse); C6, H4-II-E-C3 (rat)

  • Tissues: Human tonsil, prostate cancer tissue

  • Recombinant HMGB1 protein for Western blot ladder control

• Negative controls:

  • Isotype-matched control antibodies

  • Secondary antibody-only controls

  • HMGB1 knockout or knockdown samples when available

  • Peptide competition controls using the immunizing peptide

• In vivo experiment controls:

  • Isotype-matched IgG at equivalent dose (e.g., 20 μg mouse IgG)

  • Vehicle control (e.g., PBS alone)

  • Untreated disease model controls

• Technical controls:

  • Loading controls for Western blot (e.g., β-actin, GAPDH)

  • Multiple antibodies targeting different HMGB1 epitopes

  • Known inducers of HMGB1 release (e.g., LPS stimulation)

• Sample processing controls:

  • Nuclear vs. cytoplasmic fractionation controls

  • Consistent sample collection and processing protocols

How can I block HMGB1 activity in functional assays?

Several approaches can effectively block HMGB1 activity in functional assays:

• Neutralizing antibodies:

  • DPH1.1 mAb blocks HMGB1-elicited cell migration in trans-well migration assays

  • Anti-HMGB1 monoclonal antibodies can block HMGB1-mediated effects in EAE models

  • Optimal antibody concentrations must be determined empirically

• In vivo blocking:

  • Intraperitoneal injection of anti-HMGB1 monoclonal antibody (20 μg for 5 consecutive days) ameliorated EAE

  • Intravenous administration of DPH1.1 mAb (220 μg/mouse) blocks recruitment of inflammatory cells to sites of necrosis and infection

• Domain-specific blocking:

  • The B box of HMGB1 mediates its cytokine activity

  • Antibodies targeting the B box may be more effective at blocking inflammatory effects

  • A box peptides can competitively inhibit HMGB1 activity

• Receptor blocking:

  • HMGB1 signals through RAGE, TLR2, and TLR4

  • Blocking these receptors can inhibit HMGB1 signaling indirectly

• Readout measurements:

  • Cell migration assays can measure HMGB1-elicited chemotaxis

  • Cytokine production (IL-4, IL-6, IL-10, IL-17, IFN-γ, TNF-α) can be measured to assess inflammatory effects

  • Clinical scores in disease models provide functional readouts

• Controls:

  • Include isotype-matched control antibodies

  • Test multiple antibody concentrations to establish dose-response relationships

  • Use recombinant HMGB1 as a positive control for activity