Hadhb Antibody

What is HADHB and why is it important in research applications?

HADHB is the beta subunit of the mitochondrial trifunctional enzyme that catalyzes the last three reactions of the mitochondrial beta-oxidation pathway, which is the major energy-producing process in tissues. This pathway breaks down fatty acids into acetyl-CoA through four consecutive reactions . HADHB has several alternative names including TP-beta, Acetyl-CoA acyltransferase, and Beta-ketothiolase . It has a calculated molecular weight of 51 kDa, though the observed molecular weight in experimental conditions ranges from 47-52 kDa depending on the antibody used .

The protein is critical in research focusing on:

• Mitochondrial energy metabolism

• Fatty acid oxidation disorders

• Metabolic diseases

• Cancer metabolism

• Adipocyte regulation

Mutations in the HADHB gene can cause mitochondrial trifunctional protein (MTP) deficiency, a serious disorder affecting fatty acid metabolism that can manifest with peripheral neuropathy and brain dysfunction .

How should I select between monoclonal and polyclonal HADHB antibodies?

The choice between monoclonal and polyclonal antibodies should be based on your specific experimental requirements:

Monoclonal antibodies (e.g., 67967-1-Ig):

• Provide higher specificity for a single epitope

• Offer greater consistency between batches

• Recommended dilution for WB: 1:5000-1:50000

• Ideal for quantitative analysis and applications requiring high reproducibility

• Shows reactivity with human, mouse, rat, and pig samples

Polyclonal antibodies (e.g., 29091-1-AP):

• Recognize multiple epitopes of HADHB

• Generally provide higher sensitivity

• Recommended dilution for WB: 1:1000-1:8000

• Better for detecting denatured proteins or in applications where the epitope might be partially masked

• Shows reactivity with human, mouse, and rat samples

For novel research questions or when working with challenging samples, begin with a well-validated polyclonal antibody. For precise quantification or when absolute specificity is essential, a characterized monoclonal antibody may be preferable.

What criteria should be considered for validating a new HADHB antibody?

Proper antibody validation is essential for reliable research results. When validating a HADHB antibody:

• Reactivity verification:

  • Test the antibody in positive control samples with known HADHB expression (heart, liver tissues)

  • The search results show positive WB detection in tissues including heart (human, mouse, rat, rabbit, pig), liver, and cell lines like HeLa, HepG2, and LNCaP

• Molecular weight confirmation:

  • Verify that the observed molecular weight matches expectations (47-52 kDa)

  • The calculated molecular weight is 51 kDa, while observed weights are 47 kDa (67967-1-Ig) and 52 kDa (29091-1-AP)

• Knockdown/knockout validation:

  • Use HADHB knockdown or knockout samples as negative controls

  • Some antibodies are already validated in KD/KO applications as mentioned in search result #6

• Cross-application testing:

  • Confirm detection across multiple techniques (WB, IHC, IF)

  • Compare results between different antibodies targeting distinct HADHB epitopes

• Expression correlation:

  • Correlate protein detection with mRNA expression (qRT-PCR)

  • Search result #8 describes qRT-PCR methods for HADHB gene expression analysis

What are the optimal conditions for Western blot analysis using HADHB antibodies?

For optimal Western blot results with HADHB antibodies:

Sample preparation:

• Extract proteins from tissues/cells using RIPA lysis buffer with protease inhibitor cocktail

• Determine protein concentration using BCA assay

• Adjust all samples to equal protein concentration

Electrophoresis and transfer conditions:

• Separate proteins using standard SDS-PAGE (suggested electrophoresis: 80V for 90 min)

• Transfer to PVDF membrane (suggested: 300 mA for 120 min)

Antibody incubation:

• Block membrane with 5% skim milk or BSA in TBST for 90 minutes

• Dilute primary antibodies:

  • Monoclonal (67967-1-Ig): 1:5000-1:50000

  • Polyclonal (29091-1-AP): 1:1000-1:8000

• Incubate with primary antibody overnight at 4°C

• Wash thoroughly with TBST

• Incubate with appropriate HRP-conjugated secondary antibody for 1-2 hours at room temperature

• Wash thoroughly with TBST

Detection and analysis:

• Visualize using chemiluminescence detection system

• Analyze band intensity using image analysis software like ImageJ

The expected molecular weight for HADHB is 47-52 kDa, with slight variations depending on the specific antibody and experimental conditions .

How should I perform immunohistochemistry with HADHB antibodies?

For immunohistochemistry applications with HADHB antibodies:

Tissue preparation:

• Fix tissues in formalin

• Embed in paraffin

• Section to 3-μm thickness

• Heat sections at 60°C for 2 hours

• Dewax in xylene and rehydrate through graded ethanol series

Antigen retrieval:

• For polyclonal antibody 29091-1-AP: Use TE buffer pH 9.0 (alternate: citrate buffer pH 6.0)

• Heat with buffer for 30 minutes

Blocking and antibody incubation:

• Block endogenous peroxidase with 3% H₂O₂ for 15 minutes

• Block non-specific binding with 5% BSA

• Dilute HADHB antibody 29091-1-AP at 1:500-1:2000

• Incubate overnight at 4°C

• Incubate with HRP-labeled secondary antibody for 1 hour at 25°C

Visualization:

• Develop with diaminobenzidine (DAB)

• Counterstain nuclei with hematoxylin

• Visualize and photograph using a microscope

Controls:

• Negative control: Incubate sections with PBS instead of primary antibody

• Positive control: Use tissues with known HADHB expression (heart, liver)

Validated tissues for IHC include human stomach tissue, human colon, and human colon cancer tissue .

What protocols should be followed for immunofluorescence/immunocytochemistry?

For immunofluorescence detection of HADHB:

Cell preparation:

• Culture cells on coverslips or appropriate chambers

• Fix cells (typically with 4% paraformaldehyde)

• Permeabilize with 0.1-0.5% Triton X-100

• Block with appropriate blocking buffer (5% BSA recommended)

Antibody incubation:

• Dilute monoclonal antibody 67967-1-Ig at 1:200-1:800 for IF/ICC

• Incubate overnight at 4°C

• Wash thoroughly with PBS

• Incubate with fluorophore-conjugated secondary antibody

• Wash thoroughly with PBS

Nuclear counterstaining and mounting:

• Counterstain nuclei with DAPI or similar DNA dye

• Mount with anti-fade mounting medium

Controls and validation:

• HeLa cells have been validated for positive IF/ICC detection with HADHB antibody 67967-1-Ig

• Consider co-staining with mitochondrial markers to confirm mitochondrial localization

For mitochondrial proteins like HADHB, ensure adequate permeabilization as the double membrane structure of mitochondria can limit antibody access.

How can I address non-specific binding or high background in Western blots?

When experiencing high background or non-specific binding with HADHB antibodies:

Antibody dilution optimization:

• Increase dilution factor within recommended ranges:

  • For monoclonal 67967-1-Ig: Try higher dilutions (1:20000-1:50000)

  • For polyclonal 29091-1-AP: Try higher dilutions (1:5000-1:8000)

Blocking improvements:

• Extend blocking time (2 hours at room temperature or overnight at 4°C)

• Test alternative blocking agents (5% BSA vs. 5% non-fat milk)

• Add 0.1-0.3% Tween-20 to blocking buffer to reduce hydrophobic interactions

Washing optimization:

• Increase number of washes (5-6 washes)

• Extend washing duration (10 minutes per wash)

• Use fresh wash buffer for each washing step

• Add detergent (0.1% Tween-20) to wash buffer

Sample preparation refinements:

• Ensure complete protein denaturation

• Add protease inhibitor cocktail during extraction

• Centrifuge samples at high speed (16,000g) to remove debris

• Filter lysates before loading

Antibody quality:

• Store antibodies according to manufacturer recommendations (-20°C, avoid freeze-thaw cycles)

• Aliquot antibodies to minimize freeze-thaw cycles

• Use freshly diluted antibody preparations

If persistent background issues occur with one antibody format, consider switching between monoclonal and polyclonal antibodies as they offer different specificity profiles.

What strategies can help optimize HADHB detection in tissues with low expression?

For tissues with low HADHB expression levels:

Sample enrichment:

• Perform subcellular fractionation to isolate mitochondria

• Use larger amounts of starting material

• Concentrate protein samples using precipitation methods

Signal amplification in Western blot:

• Use more sensitive detection systems (e.g., enhanced chemiluminescence)

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

• Reduce antibody dilution (within manufacturer's recommended range)

• Use signal enhancers compatible with your detection system

Signal amplification in IHC/IF:

• Implement tyramide signal amplification (TSA)

• Use biotin-streptavidin amplification systems

• Extend DAB development time carefully while monitoring background

• Use more concentrated primary antibody (within recommended range)

Antigen retrieval optimization for IHC:

• Test both recommended retrieval methods (TE buffer pH 9.0 and citrate buffer pH 6.0)

• Optimize retrieval duration and temperature

• Consider using pressure cooker-based retrieval methods

Alternative detection methods:

• Consider more sensitive techniques like proximity ligation assay

• Use ELISA for quantitative detection in homogenates

Why might I observe discrepancies in HADHB molecular weight across different experiments?

The observed molecular weight of HADHB can vary between experiments for several reasons:

Expected variation:

• Calculated molecular weight: 51 kDa

• Observed molecular weight: 47 kDa (67967-1-Ig) or 52 kDa (29091-1-AP)

Causes of molecular weight variation:

• Post-translational modifications:

  • Phosphorylation, acetylation, or other modifications can affect migration

  • Fatty acid metabolism enzymes like HADHB may undergo regulatory modifications

• Sample preparation differences:

  • Degree of denaturation can affect protein conformation and migration

  • Buffer composition influences protein-SDS interactions

  • Reducing agent concentration affects disulfide bond disruption

• Gel system variations:

  • Acrylamide percentage affects migration patterns

  • Buffer composition (Tris-glycine vs. Tris-tricine) influences mobility

  • Gradient vs. fixed percentage gels show different resolution patterns

• Species-specific differences:

  • Slight variations in amino acid sequence between species

  • The search results show antibodies validated across human, mouse, rat, and pig samples

• Tissue-specific processing:

  • Different tissues may express variants or process the protein differently

  • Mitochondrial import and processing can vary by tissue type

To address discrepancies, always include appropriate positive controls, use a molecular weight ladder, and maintain consistent experimental conditions for comparative studies.

How can HADHB antibodies be utilized in protein-protein interaction studies?

HADHB antibodies are valuable tools for investigating protein interactions within mitochondrial energy metabolism pathways:

Co-immunoprecipitation (Co-IP):

• Lyse cells in appropriate buffer (e.g., 10 mM Hepes-KOH, pH 7.9, 0.5% NP-40, 140 mM NaCl, 10 mM KCl, 1.5 mM MgCl₂ with protease inhibitors)

• Pre-clear lysate with protein A/G beads at 4°C for 1 hour

• Incubate pre-cleared lysate with anti-HADHB antibody and protein A/G beads at 4°C for 5 hours with rotation

• Include non-immune IgG controls processed identically

• Wash precipitates and analyze by Western blot for potential binding partners

Validated interactions:

• ERβ (estrogen receptor beta) has been shown to interact and colocalize with HADHB in mitochondria

• Other mitochondrial fatty acid oxidation enzymes may be detected as interaction partners

Reciprocal confirmation:

• Perform reverse Co-IP using antibodies against suspected interaction partners

• Confirm that HADHB can be detected in these precipitates

• Validate with multiple antibodies targeting different epitopes

Advanced interaction techniques:

• Proximity ligation assay (PLA) for visualizing interactions in situ

• FRET/BRET analysis for quantifying interactions in living cells

• Crosslinking mass spectrometry for mapping interaction interfaces

The search results provide specific methodologies for Co-IP with HADHB antibodies that can be adapted for investigating new protein-protein interactions in various research contexts .

What approaches can be used to study HADHB in disease models using antibodies?

HADHB antibodies can be instrumental in studying disease models, particularly those involving mitochondrial dysfunction:

Mitochondrial trifunctional protein deficiency:

• Use Western blot to compare HADHB expression and processing in patient samples versus controls

• Analyze specific mutations (e.g., c.1175C>T [p.A392V]) and their effects on protein expression

• Monitor changes in response to treatments (e.g., low-fat, high-carbohydrate diet, MCT diet, L-carnitine supplementation)

Cancer models:

• Perform expression analysis across cancer types and stages

• Search result #2 describes HADHB analysis in stomach adenocarcinoma (STAD)

• Correlate expression with survival data using tissue microarrays

• Use IHC to assess tissue distribution in tumor samples compared to adjacent normal tissue

• Validated for human colon cancer tissue by IHC

Metabolic disease models:

• Study HADHB expression changes in diabetes, obesity, and fatty liver disease models

• Investigate adipocyte differentiation and function in obesity models

• Search result #8 describes methods for studying HADHB in adipocyte regulation

Neurological disorder models:

• Assess HADHB expression in models of peripheral neuropathy

• Search result #3 describes a case with neuropathy and higher brain dysfunction

• Use IHC to examine HADHB distribution in neural tissues

Experimental approaches:

• Gene expression manipulation (overexpression, knockdown, knockout)

• Search result #8 details methods for HADHB overexpression and analysis

• Pharmacological interventions targeting mitochondrial function

• Metabolic challenge experiments (fasting, high-fat diet, exercise)

How can HADHB antibodies be integrated with multi-omics approaches?

Modern research increasingly combines antibody-based detection with multi-omics approaches:

Proteomics integration:

• Use HADHB antibodies for immunoprecipitation followed by mass spectrometry

• Verify proteomic hits with Western blot using HADHB antibodies

• Correlate global proteomic changes with HADHB expression levels

• Combine with post-translational modification (PTM) enrichment strategies

Transcriptomics correlation:

• Pair RNA-seq data with protein-level validation using HADHB antibodies

• Search result #8 describes transcriptome sequencing of HADHB-overexpressing cells

• Identify discordant expression patterns between mRNA and protein

• Validate key differentially expressed genes (DEGs) at protein level

Pathway analysis verification:

• Use antibodies to confirm key nodes in identified pathways

• Search result #2 describes "pathway enrichment analysis" with HADHB as target

• Verify predicted protein interactions from bioinformatic analyses

• Search result #8 mentions protein-protein interaction analysis using tools like String and Cytoscape

Metabolomics connection:

• Correlate HADHB protein levels with changes in fatty acid metabolites

• Assess impact of HADHB manipulation on global metabolic profiles

• Connect metabolite changes to protein expression using antibody detection

Integrated experimental design:

• Use consistent experimental conditions across omics platforms

• Perform time-course studies with parallel sampling for different analyses

• Incorporate antibody-based imaging to provide spatial context to omics data

Search results #2 and #8 provide specific examples of integrating HADHB antibody detection with transcriptomic and pathway analyses, offering templates for multi-omics experimental design.

What techniques can be used to study HADHB localization in mitochondria?

As a mitochondrial protein, HADHB localization requires specialized approaches:

Subcellular fractionation:

• Isolate pure mitochondrial fractions from tissues/cells

• Verify fraction purity using organelle markers

• Detect HADHB distribution across fractions by Western blot

• Compare distribution under different metabolic conditions

Super-resolution microscopy:

• Use immunofluorescence with HADHB antibodies at optimized dilutions (1:200-1:800)

• Co-stain with mitochondrial markers (TOM20, MitoTracker)

• Apply techniques like STED, STORM, or SIM for sub-mitochondrial resolution

• Quantify distribution patterns using specialized image analysis software

Electron microscopy:

• Perform immunogold labeling with HADHB antibodies

• Visualize precise submitochondrial localization

• Determine association with mitochondrial cristae, matrix, or membranes

Import and processing studies:

• Compare precursor and mature forms using Western blot

• Track import kinetics using pulse-chase experiments

• Analyze processing using mitochondrial import inhibitors

Dynamic localization:

• Study redistribution under metabolic stress conditions

• Investigate co-localization with other beta-oxidation enzymes

• Examine potential mitochondrial subdomains specialized for fatty acid oxidation

The search results confirm HADHB is primarily a mitochondrial protein, with antibodies validated for detecting this localization in various experimental systems .

How can HADHB antibodies be used to investigate fatty acid metabolism?

HADHB is central to mitochondrial fatty acid beta-oxidation, making its antibodies valuable for metabolism studies:

Expression correlation with metabolic state:

• Compare HADHB protein levels across tissues with different metabolic profiles

• Analyze expression changes during fasting/feeding cycles

• Examine regulation during exercise or cold exposure

• Study developmental changes in expression patterns

Metabolic challenge experiments:

• Use high-fat diet models to study adaptive responses

• Analyze changes during fasting or caloric restriction

• Examine cold adaptation, which increases fatty acid oxidation

• Study responses to specific fatty acid types (saturated vs. unsaturated)

Functional complex assembly:

• Investigate interaction with HADHA (alpha subunit) using co-immunoprecipitation

• Study complex formation under different metabolic conditions

• Analyze defects in assembly in disease models

Disease model applications:

• Study HADHB expression in mitochondrial disease models

• Investigate metabolic adaptations in cancer, which often shows altered fatty acid metabolism

• Examine changes in obesity and diabetes models

• Search result #8 describes studying HADHB in adipocyte regulation

Pharmacological interventions:

• Monitor HADHB responses to drugs targeting mitochondrial function

• Study effects of fatty acid oxidation inhibitors

• Examine adaptation to metabolic modulators

Combining HADHB antibody detection with metabolic flux analysis provides comprehensive insights into fatty acid metabolism regulation under various physiological and pathological conditions.

What are the best practices for using HADHB antibodies in high-throughput screening?

For adapting HADHB antibody detection to high-throughput screening:

Microplate immunoassays:

• Develop ELISA protocols using validated HADHB antibodies

• Optimize antibody concentrations for signal-to-noise ratio

• Develop sandwich ELISA using complementary antibodies targeting different epitopes

• Validate with positive and negative controls

Automated Western blot systems:

• Adapt HADHB antibody protocols to capillary Western platforms

• Optimize antibody dilutions for automated systems

• Develop multiplex protocols to detect HADHB alongside other proteins

• Incorporate appropriate loading and normalization controls

Tissue microarray analysis:

• Use validated IHC conditions for HADHB detection (1:500-1:2000 dilution)

• Optimize staining parameters for automated systems

• Develop quantitative scoring methods for expression levels

• Include appropriate controls on each array

Cell-based high-content screening:

• Optimize immunofluorescence protocols for automated microscopy

• Develop robust image analysis pipelines for HADHB quantification

• Include co-staining with mitochondrial markers for colocalization analysis

• Validate using cells with manipulated HADHB expression

Quality control considerations:

• Include positive and negative controls on each plate/array

• Use standard curves where appropriate

• Incorporate technical and biological replicates

• Maintain consistent antibody lots for large-scale screens

These approaches enable systematic screening of HADHB expression and localization across multiple conditions, tissues, or compound treatments while maintaining data quality and reproducibility.