HINT2 Antibody

What is HINT2 and what are its key biological functions?

HINT2 is a mitochondrial enzyme belonging to the histidine triad protein superfamily that functions as a nucleotide hydrolase and transferase. It is predominantly located on the inner mitochondrial membrane and contains mitochondrial signal channel sequences . Key biological functions include:

• Hydrolysis of adenosine monophosphate linked to amino groups (AMP-pNA) with documented enzymatic parameters (kcat:0.0223 s-1; Km:128 μmol/L)

• Regulation of mitochondrial complex I activity and assembly, particularly affecting oxidative phosphorylation

• Protective role against pressure overload-induced cardiac remodeling and hypertrophy

• Function as a mitochondrial apoptotic sensitizer, with reduced expression observed in hepatocellular carcinoma

The protein contains the conserved HIT motif (His-X-His-X-His) with the middle Histidine being critical for its enzymatic activity . Its calculated molecular weight is 17 kDa (163 amino acids), which corresponds with the observed molecular weight in Western blot analyses .

What are the common applications of HINT2 antibody in research?

HINT2 antibody has several validated research applications that enable investigation of this protein across multiple experimental contexts:

The antibody has been tested for reactivity with human, mouse, and rat samples, making it versatile for studies across these species . These applications allow researchers to investigate HINT2 expression patterns, subcellular localization, and potential alterations in disease states.

What is the subcellular localization of HINT2?

HINT2 is predominantly localized to mitochondria. This localization has been verified through multiple experimental approaches:

• Immunocytochemistry studies with HINT2 antibody show mitochondrial staining patterns

• GFP-tagged HINT2 constructs (both N-terminal and C-terminal fusions) demonstrate mitochondrial localization when transfected into HEK-293 cells

• Biochemical fractionation studies confirm HINT2's presence in mitochondrial fractions

More specifically, HINT2 is located on the inner mitochondrial membrane, which positions it to influence critical mitochondrial functions including oxidative phosphorylation and apoptotic signaling .

Which tissue types express the highest levels of HINT2?

HINT2 exhibits a tissue-specific expression pattern, with highest expression levels in:

• Liver - showing predominant expression in hepatocytes

• Pancreas - with significant expression levels

This tissue distribution has been verified through:

• Quantitative PCR analysis of mRNA expression across multiple tissues

• Protein detection via immunoblotting of tissue lysates

Lower expression levels have been detected in other tissues, but liver and pancreas show markedly higher HINT2 expression. This tissue specificity suggests specialized functions in these metabolically active organs .

What are the recommended dilutions for HINT2 antibody in different applications?

Optimal dilutions for HINT2 antibody vary by application and should be determined empirically for each experimental system:

It is strongly recommended to perform a titration series when first using the antibody in a new experimental system to determine optimal signal-to-noise ratio .

What are the optimal protocols for detecting HINT2 via Western blot?

For optimal detection of HINT2 by Western blot, the following protocol elements are recommended:

• Sample preparation:

  • Lyse cells in PBS, pH 7.2, containing 1% Nonidet P-40, 0.4% sodium deoxycholate, and 1 mmol/L EDTA

  • Sonicate lysates and centrifuge to clear debris

  • Include protease inhibitors in lysis buffer to prevent degradation

• Gel electrophoresis:

  • Use 4-20% gradient gels for optimal separation

  • Load approximately 20-50 μg of total protein per lane

• Antibody incubation:

  • Dilute primary HINT2 antibody 1:1000-1:4000 in blocking buffer

  • Incubate membranes overnight at 4°C with gentle agitation

  • Use appropriate species-specific HRP-conjugated secondary antibody

• Detection:

  • Enhanced chemiluminescence (ECL) provides good sensitivity

  • Expected molecular weight: ~17 kDa

• Controls:

  • Positive control: HeLa cell lysate (confirmed to express HINT2)

  • Loading control: β-actin or GAPDH antibody after membrane stripping

This protocol has been validated for detection of HINT2 in human, mouse, and rat samples with high specificity.

How should I prepare samples for immunohistochemistry with HINT2 antibody?

For successful HINT2 immunohistochemistry, sample preparation is critical:

• Tissue fixation and processing:

  • Fix tissues in 4% paraformaldehyde or formalin

  • Paraffin embedding using standard protocols

  • Section tissues at 4-6 μm thickness

• Antigen retrieval:

  • Recommended method: Heat-induced epitope retrieval with TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • Optimal retrieval conditions should be determined empirically for each tissue type

• Blocking and antibody incubation:

  • Block with 5-10% normal serum from the same species as the secondary antibody

  • Dilute HINT2 antibody 1:50-1:500 in blocking buffer

  • Incubate sections overnight at 4°C in a humidified chamber

• Detection system:

  • Use biotin-streptavidin or polymer-based detection systems

  • DAB (3,3'-diaminobenzidine) substrate for visualization

  • Counterstain with hematoxylin for nuclear visualization

• Controls:

  • Positive control: human ovary cancer tissue

  • Negative control: omission of primary antibody

  • HINT2-expressing tissues like liver and pancreas as additional positive controls

This protocol has been validated for detection of HINT2 in paraffin-embedded tissue sections with high specificity and low background.

What controls should I include when using HINT2 antibody?

Appropriate controls are essential for validating HINT2 antibody results:

• Positive controls:

  • Cell lines: HeLa cells (confirmed to express HINT2)

  • Tissues: Liver and pancreas (high HINT2 expression)

  • Disease samples: Human ovary cancer tissue (validated for IHC)

• Negative controls:

  • Omission of primary antibody while maintaining all other steps

  • Isotype control using non-specific IgG at the same concentration

  • Pre-absorption of antibody with immunizing peptide (if available)

• Knockdown/knockout validation:

  • HINT2 siRNA or shRNA-treated samples to demonstrate specificity

  • CRISPR/Cas9-mediated HINT2 knockout cells as gold-standard negative control

  • Overexpression controls using HINT2 cDNA constructs for antibody sensitivity testing

• Technical controls:

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

  • Counterstaining in IHC/IF to identify tissue architecture

  • Multiple samples to account for biological variation

These controls help ensure that detected signals are specific to HINT2 and minimize the risk of false-positive or false-negative results.

Are there specific buffer conditions that optimize HINT2 antibody performance?

Buffer conditions significantly impact HINT2 antibody performance across different applications:

• Storage buffer:

  • PBS with 0.02% sodium azide and 50% glycerol, pH 7.3 provides optimal stability

  • Aliquoting is recommended for antibody in smaller sizes to avoid freeze-thaw cycles

• Western blot conditions:

  • Lysis buffer: PBS, pH 7.2, containing 1% Nonidet P-40, 0.4% sodium deoxycholate, and 1 mmol/L EDTA

  • Transfer buffer: Standard Tris-glycine with 20% methanol

  • Blocking buffer: 5% non-fat dry milk or BSA in TBST

• Immunohistochemistry buffers:

  • Antigen retrieval: TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0 (alternative)

  • Antibody diluent: PBS with 1% BSA or commercial antibody diluent

  • Wash buffer: PBS with 0.05-0.1% Tween-20

• Immunofluorescence buffers:

  • Fixation: 4% paraformaldehyde in PBS for 10 minutes

  • Permeabilization: 0.1-0.3% Triton X-100 in PBS

  • Blocking: 1-5% BSA with 0.1% Tween-20 in PBS

Buffer optimization may be necessary for each specific experimental system to achieve optimal signal-to-noise ratio.

How can I validate the specificity of a HINT2 antibody?

Multiple approaches can be used to rigorously validate HINT2 antibody specificity:

• Molecular approaches:

  • RNA interference: Compare antibody signal in cells treated with HINT2-specific siRNA versus control siRNA

  • Gene editing: Test antibody in CRISPR/Cas9-mediated HINT2 knockout cells

  • Overexpression: Detect increased signal in cells transfected with HINT2 expression constructs

• Biochemical validation:

  • Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

  • Immunoprecipitation followed by mass spectrometry: Confirm that immunoprecipitated proteins include HINT2

  • Multiple antibodies: Use antibodies targeting different epitopes of HINT2 to confirm consistent results

• Technical validation:

  • Expected molecular weight: Confirm band at approximately 17 kDa in Western blots

  • Subcellular localization: Verify mitochondrial localization in immunofluorescence

  • Tissue expression pattern: Confirm highest expression in liver and pancreas

Combining these approaches provides comprehensive validation of antibody specificity and increases confidence in experimental results.

How can I investigate HINT2's role in mitochondrial function using antibody-based techniques?

To investigate HINT2's role in mitochondrial function, several antibody-based approaches can be employed:

• Co-localization studies:

  • Perform double immunofluorescence with HINT2 antibody and mitochondrial markers (e.g., TOMM20, COX IV)

  • Use confocal microscopy to assess precise sub-mitochondrial localization

  • Analyze co-localization with mitochondrial complex I components to explore functional interactions

• Biochemical fractionation:

  • Isolate mitochondria and sub-mitochondrial fractions (outer membrane, intermembrane space, inner membrane, matrix)

  • Perform Western blotting with HINT2 antibody on each fraction

  • Compare with markers for each mitochondrial compartment to confirm inner membrane localization

• Protein-protein interaction analysis:

  • Immunoprecipitate HINT2 and analyze co-immunoprecipitated proteins by Western blot

  • Look specifically for interactions with mitochondrial complex I components (NDUF proteins)

  • Perform proximity ligation assays to detect in situ interactions with potential partners

• Functional correlations:

  • Combine HINT2 immunodetection with assessment of mitochondrial membrane potential (e.g., JC-1 staining)

  • Correlate HINT2 expression levels with markers of apoptosis and oxidative phosphorylation

  • Compare control cells with those treated with mitochondrial complex I inhibitors like rotenone

These approaches can provide insights into how HINT2 contributes to mitochondrial function and identify potential mechanism-based therapeutic interventions.

What strategies can I use to study HINT2's relationship with cardiac remodeling?

To investigate HINT2's role in cardiac remodeling, consider these antibody-based research strategies:

• Expression analysis in cardiac disease models:

  • Compare HINT2 protein levels in normal versus hypertrophic or failing hearts using Western blot

  • Perform IHC to assess spatial distribution of HINT2 in cardiac tissue sections

  • Correlate HINT2 expression with markers of cardiac hypertrophy (ANP, BNP) and remodeling

• Mechanistic studies:

  • Analyze HINT2 expression in cardiomyocytes treated with hypertrophy-inducing agents (e.g., Angiotensin II)

  • Investigate relationship between HINT2 and mitochondrial complex I components in cardiac tissue

  • Examine correlation between HINT2 levels and oxidative phosphorylation efficiency

• Genetic manipulation approaches:

  • Study cardiac phenotypes in HINT2-overexpressing or HINT2-deficient mouse models

  • Assess cardiac function parameters (echocardiography) and correlate with HINT2 protein levels

  • Analyze cardiac response to pressure overload (e.g., aortic banding) in relation to HINT2 expression

• Human studies:

  • Compare HINT2 expression in human DCM heart tissues versus normal hearts

  • Correlate HINT2 levels with clinical parameters and outcome measures

  • Investigate potential prognostic value of HINT2 expression in heart failure patients

These approaches can help establish the protective role of HINT2 in cardiac remodeling and identify potential therapeutic strategies for heart failure.

How might HINT2 antibody be used to examine its down-regulation in cancer contexts?

HINT2 antibody can be employed in multiple ways to study its down-regulation in cancer:

• Expression profiling across cancer types:

  • Perform Western blot analysis on diverse tumor tissues compared to matched normal tissues

  • Create tissue microarrays for high-throughput IHC screening of HINT2 expression

  • Focus particularly on hepatocellular carcinoma, where HINT2 down-regulation has been documented

• Correlation with clinical parameters:

  • Analyze HINT2 expression in relation to tumor grade, stage, and patient survival

  • Perform multivariate analysis to determine if HINT2 is an independent prognostic factor

  • Investigate association between HINT2 levels and response to specific therapies

• Mechanistic investigations:

  • Combine HINT2 immunodetection with markers of apoptosis (cleaved caspases, PARP)

  • Assess relationship between HINT2 expression and mitochondrial function in cancer cells

  • Study correlation between HINT2 levels and cancer cell proliferation or invasion capacity

• Experimental models:

  • Compare tumor growth in xenograft models using cancer cells with normal versus reduced HINT2 expression

  • Use immunohistochemistry to assess HINT2 expression and apoptotic markers in resulting tumors

  • Evaluate potential of HINT2 as a therapeutic target by manipulating its expression

These approaches can provide insights into how HINT2 down-regulation contributes to cancer progression and potentially identify new therapeutic strategies.

What approaches can detect interactions between HINT2 and mitochondrial complex I?

To investigate interactions between HINT2 and mitochondrial complex I components, consider these methodologies:

• Co-immunoprecipitation strategies:

  • Immunoprecipitate HINT2 from mitochondrial fractions and probe for complex I components (NDUF proteins)

  • Perform reverse co-IP using antibodies against complex I subunits and detect HINT2

  • Use crosslinking approaches to stabilize transient interactions before immunoprecipitation

• Proximity-based detection methods:

  • Proximity ligation assay (PLA) to visualize in situ interactions between HINT2 and complex I components

  • FRET (Fluorescence Resonance Energy Transfer) using fluorescently-tagged proteins

  • BioID or APEX proximity labeling to identify proteins in close proximity to HINT2 in mitochondria

• Functional correlation studies:

  • Assess impact of HINT2 manipulation (overexpression/knockdown) on complex I activity and assembly

  • Use blue native PAGE to analyze complex I assembly in relation to HINT2 expression

  • Combine with functional readouts like oxygen consumption rate and mitochondrial membrane potential

• Structural approaches:

  • Perform immunogold electron microscopy to visualize HINT2 localization relative to complex I

  • Use super-resolution microscopy techniques (STORM, PALM) for nanoscale co-localization analysis

  • Investigate binding domains through mutation studies and domain-specific antibodies

These approaches can elucidate how HINT2 influences mitochondrial complex I function and identify potential therapeutic targets for diseases involving mitochondrial dysfunction.

How can I simultaneously study HINT2 expression and apoptotic markers?

Several techniques enable simultaneous assessment of HINT2 and apoptotic markers:

• Dual immunofluorescence/immunohistochemistry:

  • Perform double immunostaining with HINT2 antibody and antibodies against apoptotic markers

  • Use different fluorophores or chromogens to distinguish signals

  • Key apoptotic markers to consider: cleaved caspase-3, cleaved caspase-9, cleaved PARP, cytochrome c

• Sequential or multiplex Western blotting:

  • Run multiple gels in parallel or use stripping and reprobing techniques

  • Employ multiplex fluorescent Western blot systems for simultaneous detection

  • Compare HINT2 levels with apoptotic marker expression under various conditions

• Flow cytometry approaches:

  • Combine intracellular staining for HINT2 with annexin V/PI for apoptosis detection

  • Include additional markers like active caspase-3 for apoptotic pathway assessment

  • Correlate HINT2 expression levels with apoptotic profiles at single-cell resolution

• Experimental manipulation:

  • Induce apoptosis with stimuli like Fas antibody (100 ng/mL) and actinomycin D (50 ng/mL)

  • Compare response in cells with normal versus altered HINT2 expression

  • Monitor impact of mitochondrial complex I inhibition on HINT2-mediated apoptotic sensitivity

These approaches can reveal how HINT2 expression correlates with and potentially regulates apoptotic pathways in normal and disease states.

Why might I observe discrepancies between HINT2 protein and mRNA levels?

Discrepancies between HINT2 protein and mRNA levels can arise from several biological and technical factors:

• Post-transcriptional regulation:

  • microRNA-mediated repression of HINT2 translation

  • RNA-binding proteins affecting mRNA stability or translation efficiency

  • Alternative splicing generating isoforms not detected by certain primers or antibodies

• Post-translational modifications and protein stability:

  • Variations in HINT2 protein half-life under different conditions

  • Proteasomal degradation pathways affecting protein but not mRNA levels

  • Post-translational modifications affecting antibody epitope recognition

• Methodological considerations:

  • Different sensitivities of protein versus mRNA detection methods

  • Sample preparation artifacts affecting protein extraction efficiency

  • Primer design for qPCR not capturing all transcript variants

• Disease-specific mechanisms:

  • In cardiac remodeling, HINT2 shows decreased protein levels in DCM hearts and hypertrophic cardiomyocytes

  • Hepatocellular carcinoma exhibits reduced HINT2 mRNA expression correlating with poor survival

  • These context-specific regulations may involve different mechanisms

When encountering such discrepancies, consider time-course experiments and inhibitors of transcription or translation to determine whether differences arise from altered synthesis or degradation rates.

What could cause inconsistent HINT2 antibody staining in immunohistochemistry?

Several factors can contribute to inconsistent HINT2 antibody staining in immunohistochemistry:

• Tissue processing and fixation issues:

  • Overfixation can mask epitopes through excessive protein crosslinking

  • Delayed fixation can lead to protein degradation and reduced signal

  • Variations in processing between samples affect antibody penetration and binding

• Antigen retrieval challenges:

  • Insufficient heat-induced epitope retrieval (HIER)

  • Incorrect pH of retrieval buffer (optimal: TE buffer pH 9.0)

  • Inconsistent retrieval conditions between experiments

• Antibody-related factors:

  • Batch-to-batch variations in antibody production

  • Inappropriate antibody dilution (recommended: 1:50-1:500)

  • Antibody degradation due to improper storage or handling

• Biological variations:

  • Natural heterogeneity in HINT2 expression across cell types within a tissue

  • Disease state affecting HINT2 expression or localization

  • Variations in mitochondrial content or integrity between samples

To address these issues, standardize all steps of the IHC protocol, include positive and negative controls with each experiment, and consider using automated IHC platforms for improved consistency.

How can I address high background when using HINT2 antibody in immunofluorescence?

High background in HINT2 immunofluorescence can be mitigated through several optimization strategies:

• Blocking optimization:

  • Increase blocking time (1-2 hours) and concentration (3-5% BSA or normal serum)

  • Include 0.1-0.3% Triton X-100 in blocking buffer to reduce non-specific binding

  • Consider adding 0.1% gelatin or 5% milk as alternative blocking agents

• Antibody dilution and incubation:

  • Test a broader dilution range (start with 1:100-1:500) to identify optimal concentration

  • Extend primary antibody incubation to overnight at 4°C

  • Increase washing steps (5-6 washes of 5-10 minutes each) after antibody incubations

• Sample preparation refinements:

  • Optimize fixation time to prevent over-fixation (typically 10-15 minutes in 4% PFA)

  • Ensure complete permeabilization for mitochondrial antigen access

  • Consider low auto-fluorescence mounting media and slides

• Controls and troubleshooting:

  • Include secondary-only controls to identify non-specific secondary antibody binding

  • Use HINT2-negative samples or HINT2-knockdown cells as negative controls

  • Consider pre-absorption of antibody with immunizing peptide to block specific binding

If background persists despite these optimizations, consider alternative detection methods like tyramide signal amplification, which can allow more dilute primary antibody use while maintaining signal intensity.

What approaches can help resolve non-specific bands in HINT2 Western blots?

To address non-specific bands in HINT2 Western blots, consider these optimization strategies:

• Sample preparation refinements:

  • Include additional protease inhibitors in lysis buffer

  • Optimize protein extraction conditions (buffer composition, sonication parameters)

  • Perform subcellular fractionation to enrich for mitochondrial proteins

• Gel electrophoresis and transfer optimization:

  • Increase gel percentage (12-15%) to better resolve proteins near 17 kDa

  • Optimize transfer conditions (time, voltage, buffer composition)

  • Consider gradient gels (4-20%) for better resolution across a wider range of molecular weights

• Antibody optimization:

  • Test multiple dilutions to identify optimal signal-to-noise ratio (1:1000-1:4000)

  • Increase washing time and frequency after antibody incubations

  • Consider alternative blocking agents (5% BSA instead of milk for phospho-specific antibodies)

• Validation approaches:

  • Run positive control (HeLa cell lysate) alongside experimental samples

  • Include samples with HINT2 knockdown or overexpression as controls

  • Use HINT2 recombinant protein as size reference and positive control

If a band at the expected size (~17 kDa) is consistently present along with non-specific bands, consider using more stringent washing conditions or testing a different HINT2 antibody that recognizes a different epitope.

How should I interpret changes in HINT2 expression in disease models?

When interpreting alterations in HINT2 expression in disease models, consider these analytical approaches:

• Context-specific biological significance:

  • In cardiac remodeling: Decreased HINT2 correlates with increased hypertrophy markers like ANP

  • In hepatocellular carcinoma: Reduced HINT2 associates with poor survival

  • Changes should be interpreted in relation to established disease markers and functional readouts

• Correlation with functional parameters:

  • Relate HINT2 expression to mitochondrial function (membrane potential, oxygen consumption)

  • Assess relationship with apoptotic sensitivity and markers (cleaved caspases, PARP)

  • Examine impact on tissue-specific functions (cardiac contractility, hepatic metabolism)

• Mechanistic considerations:

  • Determine whether changes are primary disease drivers or secondary adaptations

  • Examine relationship with known disease pathways (e.g., mitochondrial complex I in cardiac disease)

  • Consider temporal dynamics of expression changes throughout disease progression

• Translational implications:

  • Evaluate potential of HINT2 as a diagnostic or prognostic biomarker

  • Consider therapeutic implications of restoring normal HINT2 levels

  • Assess HINT2 expression in response to current therapeutic interventions

Rigorous quantification and appropriate statistical analysis are essential when comparing HINT2 expression between experimental groups. Changes should be reproducible across multiple experimental models and methodologies to establish robust disease associations.