hdhd2 Antibody

What is HDHD2 and why is it significant for research studies?

HDHD2 (Haloacid Dehalogenase-Like Hydrolase Domain Containing 2) is a 259 amino acid protein expressed as two isoforms produced by alternative splicing . This enzyme belongs to the haloacid dehalogenase-like hydrolase domain family and is encoded by the HDHD2 gene located on human chromosome 18 .

Research significance:

• Functions in enzyme binding activities and potential phosphatase activity

• Involved in dephosphorylation and potentially protein transport

• Associated with diseases including Chromosome 18q Deletion Syndrome

• Expressed in multiple tissues with observed molecular weights of 18 kDa and 28 kDa in experimental contexts

For reproducible Western blot detection of HDHD2:

• Sample preparation protocol:

  • Harvest cells or homogenize tissue in RIPA buffer containing protease inhibitors

  • Centrifuge lysate at 12,000g for 15 minutes at 4°C

  • Determine protein concentration using Bradford or BCA assay

  • Prepare 20-30 μg protein samples in Laemmli buffer with reducing agent

• Electrophoresis considerations:

  • Use 10-12% SDS-PAGE gels for optimal resolution of HDHD2

  • Include positive controls (HepG2 cells or mouse liver tissue)

  • HDHD2 may be observed at different molecular weights (18 kDa and 28 kDa)

• Transfer and detection:

  • Transfer to PVDF membrane at 100V for 60-90 minutes

  • Block with 5% non-fat milk in TBST for 1 hour

  • Incubate with primary HDHD2 antibody at 1:500-1:2000 dilution overnight at 4°C

  • Wash membrane 3x with TBST and incubate with appropriate HRP-conjugated secondary antibody

  • Develop using enhanced chemiluminescence

How do monoclonal and polyclonal HDHD2 antibodies compare in terms of specificity and sensitivity?

A comparative analysis of HDHD2 antibody types reveals important considerations for experimental design:

Monoclonal HDHD2 antibodies:

• Higher specificity with reduced cross-reactivity

• Examples include mouse monoclonal clones OTI1A5 and OTI2C12

• Optimal for applications requiring high reproducibility and minimal batch variation

• Validated applications include WB (1:2000), IHC (1:150), and FC (1:100)

• Typically generated using full-length human recombinant HDHD2 protein produced in HEK293T cells

Polyclonal HDHD2 antibodies:

• Recognize multiple epitopes, potentially increasing detection sensitivity

• Available from rabbit hosts with various immunogens (full length protein or specific amino acid regions)

• Different polyclonal products target different regions (AA 1-259, AA 97-249, AA 1-100)

• May show higher background in some applications

• Useful for detecting proteins in native conformation or proteins with post-translational modifications

Selection criteria should be based on specific experimental requirements, with monoclonal antibodies preferred when absolute specificity is critical, and polyclonal antibodies when sensitivity and detection of modified forms is prioritized.

What factors contribute to inconsistent HDHD2 detection across different experimental contexts?

Several technical and biological factors may explain variability in HDHD2 detection:

• Post-translational modifications:

  • HDHD2 may undergo modification affecting antibody recognition

  • Different antibodies may have varying sensitivity to modified epitopes

• Isoform specificity:

  • HDHD2 is expressed as two isoforms from alternative splicing

  • Antibodies targeting specific regions may not detect all isoforms

  • For comprehensive detection, use antibodies validated against multiple isoforms

• Sample preparation variables:

  • Fixation protocols significantly impact epitope accessibility in IHC/IF

  • For formalin-fixed samples, antigen retrieval optimization is critical

  • Cell lysis conditions affect protein solubilization and epitope exposure

• Technical considerations:

  • Observed molecular weights vary (18 kDa, 28 kDa, 32 kDa) across studies

  • Storage conditions affect antibody performance (store at -20°C with glycerol)

  • Different detection systems (ECL vs. fluorescent) have varying sensitivity thresholds

To address these variables, researchers should include appropriate positive controls (HepG2 cells or mouse liver tissue) and validate new applications with multiple antibodies when possible.

How can researchers validate HDHD2 antibody specificity in their experimental system?

A comprehensive validation strategy for HDHD2 antibodies includes:

• Positive and negative control samples:

  • Positive controls: HepG2 cells and mouse liver tissue show detectable HDHD2 expression

  • Negative controls: Secondary antibody-only controls to assess background

  • Tissue panel testing to confirm expected expression patterns

• Knockdown/knockout validation:

  • siRNA or CRISPR-mediated HDHD2 knockdown/knockout

  • Demonstrate reduction/absence of signal in Western blot and immunostaining

  • Include scrambled siRNA controls to confirm specificity

• Recombinant protein controls:

  • Use recombinant HDHD2 protein as positive control

  • Test antibody against related family members to assess cross-reactivity

  • Consider testing against LHPP, an important paralog of HDHD2

• Cross-validation with multiple antibodies:

  • Compare results using antibodies targeting different epitopes

  • Confirm consistent detection pattern across polyclonal and monoclonal antibodies

  • Evaluate concordance between protein and mRNA expression data

• Mass spectrometry validation:

  • Confirm antibody-detected bands by immunoprecipitation followed by mass spectrometry

  • Compare detected proteins against HDHD2 sequence database

What are the most effective protocols for HDHD2 immunohistochemical detection?

For optimized IHC detection of HDHD2:

• Sample preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard protocols

  • Section at 4-5 μm thickness

• Antigen retrieval methods:

  • Heat-induced epitope retrieval: Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Pressure cooker treatment: 125°C for 3 minutes or 95°C for 20 minutes

  • Allow slides to cool to room temperature gradually (20 minutes)

• Staining protocol optimization:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Protein block: 5% normal serum for 30 minutes

  • Primary antibody incubation: HDHD2 antibody at 1:100-1:200 dilution

  • Optimal incubation conditions: overnight at 4°C or 60 minutes at room temperature

  • Detection system: Use polymer-based detection systems for improved sensitivity

  • Counterstain with hematoxylin for 30 seconds

• Controls and validation:

  • Include tissue known to express HDHD2 as positive control

  • Use isotype control at the same concentration as primary antibody

  • Consider dual staining with markers of subcellular compartments

The recommended dilution range for IHC applications is 1:100-1:200, but this should be optimized for each specific antibody and tissue type .

How can researchers troubleshoot weak or absent HDHD2 signal in Western blot applications?

Common challenges and solutions for HDHD2 Western blotting:

• Weak or no signal:

  • Increase protein loading (30-50 μg)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize antibody concentration (try range from 1:500 to 1:2000)

  • Use enhanced sensitivity detection reagents

  • Check tissue/cell expression levels (HepG2 cells show good expression)

• Multiple bands or unexpected molecular weight:

  • HDHD2 may appear at different molecular weights (18 kDa, 28 kDa, 32 kDa)

  • This may represent different isoforms or post-translational modifications

  • Use positive control lysates with known HDHD2 expression

  • Consider using fresh samples to minimize protein degradation

  • Add additional protease inhibitors to extraction buffer

• High background:

  • Increase blocking time (2 hours) and washing steps (5 × 5 minutes)

  • Use 5% BSA instead of milk for blocking and antibody dilution

  • Reduce secondary antibody concentration

  • For polyclonal antibodies, pre-adsorb against other species proteins

• Sample preparation improvements:

  • Add phosphatase inhibitors to lysis buffer

  • Optimize lysis conditions for subcellular fractionation

  • Consider using different detergents for membrane protein extraction

What are the critical factors for optimizing immunofluorescence detection of HDHD2?

For successful immunofluorescence detection of HDHD2:

• Cell preparation protocols:

  • Culture cells on sterile coverslips or chamber slides

  • Fix with 4% paraformaldehyde (10 minutes) for best epitope preservation

  • Permeabilize with 0.1-0.3% Triton X-100 for 5-10 minutes

  • Block with 5% normal serum from secondary antibody host species

• Antibody selection and optimization:

  • For IF/ICC applications, use antibodies validated for this purpose

  • Test dilution range from 1:50-1:500 to determine optimal concentration

  • Consider directly conjugated antibodies (FITC) for certain applications

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

• Imaging parameters:

  • Use appropriate excitation/emission filters for conjugates (FITC: 499/515 nm)

  • Capture z-stack images to assess subcellular localization

  • Include DAPI nuclear counterstain

  • Use confocal microscopy for higher resolution of cellular localization

• Controls and validation:

  • Include secondary-only controls

  • Compare staining pattern with published subcellular localization data

  • Use HeLa cells as positive control for HDHD2 expression

  • Consider co-staining with organelle markers to confirm subcellular localization

How can HDHD2 antibodies be used to investigate protein-protein interactions and complex formation?

Advanced methodologies for studying HDHD2 interactions:

• Co-immunoprecipitation protocols:

  • Use HDHD2 antibodies for immunoprecipitation from cell/tissue lysates

  • Cross-link antibodies to protein A/G beads to reduce IgG contamination

  • Consider mild lysis conditions to preserve protein complexes

  • Analyze precipitated complexes by Western blot or mass spectrometry

• Proximity ligation assay (PLA):

  • Detect protein interactions in situ with spatial resolution

  • Combine HDHD2 antibodies with antibodies against suspected interaction partners

  • Use species-specific secondary antibodies with oligonucleotide probes

  • Amplification creates fluorescent spots where proteins are in close proximity

• FRET/BRET analysis:

  • Tag HDHD2 and potential binding partners with appropriate fluorophores

  • Measure energy transfer as indicator of protein-protein proximity

  • Optimize donor/acceptor ratios for maximum sensitivity

• Chromatin immunoprecipitation (ChIP):

  • Investigate potential involvement of HDHD2 in chromatin regulation

  • Cross-link protein-DNA complexes and immunoprecipitate with HDHD2 antibodies

  • Analyze associated DNA by qPCR or sequencing

How do different HDHD2 antibodies perform in detecting post-translational modifications?

Post-translational modification detection strategies:

• Phosphorylation-specific detection:

  • Standard HDHD2 antibodies may not distinguish phosphorylated forms

  • Use phospho-specific antibodies if available

  • Compare band patterns with and without phosphatase treatment

  • Consider 2D gel electrophoresis to separate modified forms

• Approach for detecting other modifications:

  • Use modification-specific antibodies in combination with HDHD2 antibodies

  • Immunoprecipitate HDHD2 and probe with antibodies against modifications (ubiquitin, SUMO, etc.)

  • Mass spectrometry analysis of immunoprecipitated HDHD2 to identify modifications

• Technical considerations:

  • Include phosphatase/deubiquitinase inhibitors in lysis buffers

  • Use appropriate positive controls for each modification

  • Consider enrichment strategies for modified proteins before Western blotting

• Correlation with functional analysis:

  • Combine detection of modified forms with functional assays

  • Correlate modification status with subcellular localization

  • Investigate temporal dynamics of modifications under various conditions

What approaches can be used to study tissue-specific expression patterns of HDHD2?

Comprehensive tissue expression analysis methods:

• Tissue microarray (TMA) analysis:

  • Use HDHD2 antibodies validated for IHC applications (1:100-1:200 dilution)

  • Apply to TMAs containing multiple tissue types

  • Score expression levels using digital pathology quantification

  • Correlate with clinical parameters if using patient samples

• Multiplex immunofluorescence:

  • Combine HDHD2 antibodies with cell type-specific markers

  • Use spectrally distinct fluorophores for each antibody

  • Apply tissue clearing techniques for 3D visualization

  • Quantify co-expression patterns using advanced image analysis

• Single-cell analysis approaches:

  • Apply HDHD2 antibodies in flow cytometry (1:100 dilution)

  • Combine with cell type-specific surface markers

  • Consider mass cytometry (CyTOF) for higher dimensionality

  • Correlate protein expression with single-cell transcriptomics

• Comparative expression analysis:

  • Use standardized IHC protocols across tissue panels

  • Include antibodies against related family members for comparison

  • Apply digital pathology quantification for objective assessment

  • Compare with publicly available transcriptomic datasets

How should researchers interpret differences in HDHD2 molecular weight observed across different studies?

Analysis of molecular weight variations:

• Documented weight variations:

  • 18 kDa and 28 kDa observed in Western blots by Proteintech

  • 32 kDa reported by Abbexa

  • Predicted molecular weight of 28.4-28.5 kDa

• Biological explanations:

  • Alternative splicing produces multiple isoforms

  • Post-translational modifications may alter migration

  • Proteolytic processing could generate smaller fragments

  • Different subcellular localization or compartment-specific processing

• Technical factors:

  • Gel percentage affects protein migration

  • Sample preparation (reducing vs. non-reducing conditions)

  • Buffer composition and detergents used during extraction

  • Calibration of molecular weight markers

• Validation approaches:

  • Compare with recombinant HDHD2 protein standards

  • Perform mass spectrometry to confirm identity of bands

  • Use antibodies targeting different epitopes to verify detection

What statistical approaches are recommended for quantifying HDHD2 expression in different experimental contexts?

Robust statistical methodologies for HDHD2 quantification:

• Western blot quantification:

  • Normalize HDHD2 signal to appropriate loading controls (β-actin, GAPDH)

  • Use at least three biological replicates per condition

  • Apply ANOVA with post-hoc tests for multi-group comparisons

  • Consider non-parametric tests if normality assumptions are violated

  • Report fold-change with 95% confidence intervals

• Immunohistochemistry quantification:

  • Use digital pathology software for unbiased quantification

  • Develop standardized scoring system (H-score, Allred score)

  • Assess both staining intensity and percentage of positive cells

  • For tissue microarrays, use mixed-effects models to account for spot variability

  • Calculate intraclass correlation coefficients for inter-observer reliability

• Flow cytometry analysis:

  • Report median fluorescence intensity rather than mean

  • Use appropriate compensation controls

  • Apply non-parametric statistical tests for non-normal distributions

  • Consider dimensionality reduction techniques for complex datasets

• Image-based quantification:

  • Establish consistent thresholding parameters

  • Use nuclear counterstain for cell normalization

  • Report intensity per cell or area rather than total intensity

  • Consider machine learning approaches for complex pattern recognition

How can researchers integrate HDHD2 protein expression data with transcriptomic and functional data sets?

Multi-omics integration strategies:

• Correlation analysis approaches:

  • Calculate Pearson or Spearman correlations between protein and mRNA levels

  • Use public databases to compare your protein expression data with RNA-seq datasets

  • Consider time-course experiments to capture expression dynamics

  • Apply methods that account for different dynamic ranges of techniques

• Pathway and network analysis:

  • Map HDHD2 interactions using STRING or BioGRID databases

  • Perform gene set enrichment analysis (GSEA) with correlated genes

  • Use Cytoscape for network visualization

  • Integrate with phosphoproteomics data to identify signaling networks

• Functional data integration:

  • Correlate HDHD2 levels with enzyme activity measurements

  • Link expression patterns to phenotypic outcomes

  • Use machine learning to identify predictive signatures

  • Apply causal network modeling to infer regulatory relationships

• Visualization and reporting:

  • Create multi-panel visualizations showing protein, RNA, and functional data

  • Use dimensionality reduction methods to visualize complex relationships

  • Implement interactive dashboards for data exploration

  • Clearly report correlation coefficients with appropriate statistical significance