CHDH Antibody

What is CHDH and why is it significant in research?

CHDH (Choline Dehydrogenase) is a crucial FAD-dependent enzyme consisting of 594 amino acids that belongs to the GMC oxidoreductase family. It plays a vital role in choline metabolism, an essential micronutrient serving as a major source of methyl groups in the human diet. This enzyme catalyzes the conversion of choline to betaine aldehyde, which is subsequently oxidized to betaine, a precursor of methionine. This process influences various biological processes, including methylation reactions critical for DNA synthesis and repair. CHDH is particularly significant in research due to its potential as a biomarker for early-stage estrogen receptor-positive breast cancer and its association with varying susceptibility to choline deficiency through gene polymorphisms .

What are the primary applications for CHDH antibodies in research?

CHDH antibodies are employed across multiple experimental applications, with Western Blot (WB) being the most common. Other frequently used applications include ELISA (Enzyme-Linked Immunosorbent Assay), Immunohistochemistry (IHC), Immunoprecipitation (IP), and Immunofluorescence (IF). These applications allow researchers to detect and measure CHDH protein expression, localization, and interactions in various biological samples. The versatility of these applications makes CHDH antibodies valuable tools for investigating choline metabolism pathways, mitochondrial function, and related disease mechanisms .

What is the typical cellular localization of CHDH protein?

CHDH protein is primarily localized to the mitochondria of cells, specifically on the matrix side of the inner mitochondrial membrane. This localization is consistent with its role in cellular energy metabolism and homeostasis. The highest CHDH activity is observed in kidney and liver tissues, though it is expressed across multiple tissue types including testis. When performing immunofluorescence or immunohistochemistry experiments, researchers should expect mitochondrial staining patterns, which can be verified using co-localization with established mitochondrial markers .

How should I design a Western blot experiment to detect CHDH protein?

When designing a Western blot experiment for CHDH detection, consider the following methodological approach:

• Sample preparation: Extract proteins from tissues with known CHDH expression (kidney, liver, testis) using mitochondrial isolation protocols for enrichment.

• Electrophoresis conditions: Use SDS-PAGE with 10-12% gels, as CHDH has a molecular weight of approximately 65.4 kDa.

• Transfer conditions: Semi-dry or wet transfer at 100V for 1-2 hours is typically effective.

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody: Dilute CHDH antibody according to manufacturer's recommendation (typically 1:200-1:1000) and incubate overnight at 4°C.

• Secondary antibody: Use species-appropriate HRP-conjugated secondary antibody.

• Controls: Include positive control tissues (kidney, liver) and negative controls.

For enhanced specificity, consider using monoclonal antibodies like the mouse monoclonal IgG1 kappa light chain antibody that detects CHDH protein from mouse, rat, and human samples .

What are the optimal conditions for immunofluorescence staining with CHDH antibodies?

For optimal immunofluorescence staining of CHDH protein, follow this methodological approach:

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.2% Triton X-100.

• Blocking: 2-5% normal serum (matching secondary antibody host) with 1% BSA for 1 hour.

• Primary antibody: Dilute CHDH antibody (typically 1:50-1:200) and incubate overnight at 4°C.

• Secondary antibody: Fluorophore-conjugated secondary antibody (Alexa Fluor conjugates are ideal) at 1:500 dilution for 1 hour at room temperature.

• Counterstain: DAPI for nuclear visualization and MitoTracker for mitochondrial co-localization.

• Mounting: Anti-fade mounting medium to preserve fluorescence.

Since CHDH is primarily localized to mitochondria, a punctate cytoplasmic staining pattern should be observed, with higher intensity in tissues like kidney and liver. Using conjugated forms of CHDH antibodies (FITC, PE, or Alexa Fluor conjugates) can simplify the protocol by eliminating the need for secondary antibodies .

How can I validate the specificity of a CHDH antibody?

Validating CHDH antibody specificity requires multiple complementary approaches:

• Western blot analysis: Confirm a single band at the expected molecular weight (65.4 kDa).

• Immunoprecipitation followed by mass spectrometry: Verify the pulled-down protein is indeed CHDH.

• siRNA or CRISPR knockout validation: Demonstrate reduced or absent signal in CHDH-depleted samples.

• Peptide competition assay: Pre-incubate antibody with blocking peptide to show signal reduction.

• Tissue panel analysis: Compare expression patterns with known CHDH distribution (highest in kidney and liver).

• Cross-reactivity testing: Test against recombinant CHDH and related proteins to confirm specificity.

• Multiple antibody validation: Compare results from antibodies targeting different CHDH epitopes.

This multi-faceted approach ensures that your experimental observations are truly reflective of CHDH biology rather than non-specific interactions or artifacts .

How can CHDH antibodies be used to investigate mitochondrial dysfunction in disease models?

CHDH antibodies serve as powerful tools for investigating mitochondrial dysfunction in various disease models through several advanced methodological approaches:

• Co-immunoprecipitation studies: Use CHDH antibodies to pull down protein complexes and identify novel interaction partners that may be dysregulated in disease states.

• Proximity ligation assays: Combine CHDH antibodies with antibodies against other mitochondrial proteins to visualize and quantify protein-protein interactions in situ.

• Super-resolution microscopy: Employ fluorophore-conjugated CHDH antibodies for nanoscale localization imaging to detect subtle changes in mitochondrial organization.

• Tissue microarray analysis: Apply CHDH immunohistochemistry across disease-specific tissue microarrays to correlate expression patterns with clinical outcomes.

• Flow cytometry with mitochondrial function dyes: Combine CHDH antibody staining with mitochondrial membrane potential dyes to correlate CHDH levels with functional mitochondrial parameters.

These approaches allow researchers to connect CHDH expression and localization to broader mitochondrial health metrics, particularly relevant in diseases with known metabolic dysregulation such as neurodegenerative disorders, cancer, and cardiovascular disease .

What is the relationship between CHDH expression and estrogen signaling in breast cancer research?

The relationship between CHDH expression and estrogen signaling presents a promising area for breast cancer research, as estrogen regulates the gene encoding CHDH. This connection suggests methodological approaches for investigating CHDH as a potential biomarker for early-stage estrogen receptor-positive breast cancer, particularly in predicting anti-estrogen resistance:

• ChIP-seq analysis: Using antibodies against estrogen receptor (ER) to identify direct binding to CHDH promoter regions.

• Dual immunohistochemistry: Co-staining for CHDH and ER in breast cancer tissue microarrays to establish correlation patterns.

• Hormone manipulation studies: Measuring CHDH expression changes after estrogen stimulation or anti-estrogen treatment using quantitative immunoblotting.

• Patient-derived xenograft models: Correlating CHDH expression with tamoxifen/aromatase inhibitor response using CHDH immunohistochemistry.

• Longitudinal biomarker studies: Monitoring CHDH expression in patient samples before and during endocrine therapy resistance development.

These methodological approaches help elucidate whether CHDH expression patterns can serve as predictive biomarkers for endocrine therapy response and inform treatment decisions in estrogen receptor-positive breast cancer patients .

How can CHDH antibodies be employed in studying choline metabolism dysregulation?

CHDH antibodies provide valuable research tools for investigating choline metabolism dysregulation through several sophisticated methodological approaches:

• Metabolic flux analysis: Combining CHDH immunoprecipitation with activity assays to measure enzyme kinetics in different physiological or pathological states.

• Tissue-specific metabolome correlation: Correlating CHDH protein levels (via quantitative immunoblotting) with choline, betaine, and homocysteine measurements across tissues.

• Nutritional intervention studies: Monitoring CHDH expression and localization changes in response to varying dietary choline intake using immunohistochemistry and subcellular fractionation.

• Genetic polymorphism analysis: Correlating known CHDH polymorphisms with protein expression/function using genotype-specific tissue samples and antibody-based quantification.

• Multi-omics integration: Combining CHDH immunoprecipitation with proteomics to identify condition-specific interaction partners that influence metabolic outcomes.

These approaches help researchers understand how alterations in CHDH expression or activity contribute to metabolic dysregulation in conditions such as non-alcoholic fatty liver disease, neural tube defects, and cognitive disorders associated with choline metabolism disturbances .

How can I resolve weak or absent signal issues when using CHDH antibodies?

When encountering weak or absent signals with CHDH antibodies, consider these methodological troubleshooting approaches:

• Sample preparation optimization:

  • Use mitochondrial enrichment protocols to concentrate CHDH protein

  • Employ gentler lysis buffers to preserve protein structure

  • Avoid repeated freeze-thaw cycles of lysates

• Antibody optimization:

  • Test different concentrations (titration series from 1:50 to 1:1000)

  • Extend incubation time (overnight at 4°C)

  • Try different antibody clones targeting different epitopes

• Detection system enhancement:

  • Use signal amplification systems (TSA, polymer-based detection)

  • Employ more sensitive detection substrates for Western blot

  • Increase exposure time while monitoring background

• Protocol modifications:

  • Adjust blocking conditions (try BSA instead of milk or vice versa)

  • Modify antigen retrieval methods for fixed tissues

  • Reduce washing stringency to preserve antibody binding

• Consider tissue/sample type:

  • Test known high-expressing tissues (kidney, liver) as positive controls

  • Account for species differences in epitope conservation

  • Verify sample quality with housekeeping proteins

This systematic approach helps identify and resolve technical issues that may obscure CHDH detection across different experimental platforms .

What are common pitfalls in immunoprecipitation experiments with CHDH antibodies?

Immunoprecipitation with CHDH antibodies presents specific challenges that require methodological attention:

• Mitochondrial localization challenges:

  • Use specialized lysis buffers containing 1% digitonin or NP-40 to solubilize membrane-bound proteins

  • Consider crosslinking approaches to preserve transient interactions

  • Maintain samples at 4°C throughout to prevent protein degradation

• Antibody orientation and binding issues:

  • Use antibodies validated specifically for IP applications

  • Consider pre-clearing lysates with protein A/G beads

  • Test both direct antibody coupling to beads and indirect capture methods

• Interference complications:

  • Heavy chain interference at ~50 kDa can mask CHDH detection in the IP eluate

  • Use HRP-conjugated protein A/G or light-chain specific secondary antibodies

  • Consider native elution conditions to preserve CHDH activity for downstream analysis

• Low abundance issues:

  • Scale up starting material (especially for tissues with moderate expression)

  • Optimize antibody-to-lysate ratios

  • Consider longer incubation times (overnight at 4°C with gentle rotation)

• Validation approaches:

  • Perform reverse IP with known interaction partners

  • Include IgG control IP to identify non-specific binding

  • Confirm specific enrichment by comparing input, flow-through, and eluate fractions

These methodological considerations help maximize success when performing immunoprecipitation experiments with CHDH antibodies .

How do I select the most appropriate CHDH antibody for my specific research application?

Selecting the optimal CHDH antibody requires consideration of several methodological factors:

• Application-specific validation:

  • For Western blot: Prioritize antibodies with demonstrated single-band specificity at 65.4 kDa

  • For IHC/IF: Select antibodies validated in fixed tissues with appropriate controls

  • For IP: Choose antibodies specifically validated for immunoprecipitation applications

  • For flow cytometry: Ensure antibodies work in appropriate fixation/permeabilization conditions

• Clone type considerations:

  • Monoclonal antibodies (like CHDH Antibody C-5) offer high specificity but limited epitope recognition

  • Polyclonal antibodies provide broader epitope recognition but potential batch variation

  • Recombinant antibodies offer consistency across experiments

• Species reactivity requirements:

  • Verify cross-reactivity with your experimental species (human, mouse, rat)

  • Consider epitope conservation across species if working with unusual model organisms

• Conjugation needs:

  • Direct conjugates (HRP, PE, FITC, Alexa Fluor) eliminate secondary antibody requirements

  • Unconjugated antibodies offer flexibility across applications but require secondary detection

• Technical specifications review:

  • Evaluate titration data across relevant dilution ranges

  • Assess positive control tissue data (kidney, liver should show strong signals)

  • Review literature citations using the specific antibody clone

This methodological framework ensures selection of the most appropriate CHDH antibody for your specific research needs, enhancing experimental success and data reliability .

How can CHDH antibodies be integrated into single-cell analysis workflows?

Integrating CHDH antibodies into single-cell analysis workflows requires specialized methodological approaches:

• Single-cell Western blot/capillary electrophoresis:

  • Optimize CHDH antibody dilutions for reduced sample volumes

  • Use signal amplification systems to detect potentially low abundance in individual cells

  • Normalize against mitochondrial content markers for accurate comparisons

• Mass cytometry (CyTOF) integration:

  • Conjugate CHDH antibodies with rare metal isotopes

  • Validate metal-tagged antibodies against fluorophore counterparts

  • Combine with mitochondrial health markers and metabolic enzyme panels

• Imaging mass cytometry applications:

  • Optimize CHDH antibodies for tissue section compatibility

  • Establish appropriate antigen retrieval methods

  • Develop multiplexed panels including metabolic and cell type markers

• Single-cell proteomics workflows:

  • Use CHDH antibodies for sorting based on expression levels

  • Incorporate CHDH into antibody barcoding strategies

  • Validate capture efficiency in microfluidic systems

• Spatial transcriptomics correlation:

  • Combine CHDH immunostaining with spatial transcriptomics

  • Correlate protein expression with mRNA levels at single-cell resolution

  • Map mitochondrial function heterogeneity across tissue regions

These advanced methodological applications allow researchers to investigate cell-to-cell variability in CHDH expression and its relationship to mitochondrial function and choline metabolism at unprecedented resolution .

What role can computational antibody design play in developing next-generation CHDH antibodies?

Computational antibody design represents a frontier for developing next-generation CHDH antibodies with enhanced specificity and functionality:

• Epitope-focused design strategies:

  • Analyze CHDH protein structure to identify accessible, unique epitopes

  • Design antibodies targeting functionally relevant domains (FAD-binding region, catalytic site)

  • Create antibodies distinguishing between CHDH conformational states

• Implementation of AI-based approaches:

  • Utilize fine-tuned RFdiffusion networks to generate antibody structures with atomic-level precision

  • Design complementarity-determining regions (CDRs) with optimal binding energetics

  • Perform in silico affinity maturation to enhance binding properties

• Experimental validation pathway:

  • Screen computational designs using yeast display technologies

  • Characterize binding properties using multiple biophysical methods

  • Verify structural accuracy through cryo-EM or X-ray crystallography

• Function-specific antibody development:

  • Design antibodies that distinguish active vs. inactive CHDH conformations

  • Create antibodies specific to post-translationally modified forms

  • Develop antibodies targeting CHDH-protein interaction interfaces

• Practical applications:

  • Design species-specific antibodies for comparative metabolism studies

  • Create antibodies with improved tissue penetration for in vivo imaging

  • Develop therapeutic antibodies modulating CHDH activity in disease contexts

This cutting-edge methodological approach represents the future of CHDH antibody development, promising reagents with precisely tailored properties for specialized research applications .

How might CHDH antibodies contribute to understanding neurodegenerative disease mechanisms?

CHDH antibodies offer valuable methodological approaches for investigating neurodegenerative disease mechanisms:

• Brain region-specific analysis:

  • Apply CHDH immunohistochemistry across brain regions affected in different neurodegenerative diseases

  • Quantify region-specific changes in CHDH expression relative to disease progression

  • Correlate with markers of neuronal health and mitochondrial function

• Cellular models of neurodegeneration:

  • Monitor CHDH expression and localization in induced pluripotent stem cell (iPSC)-derived neurons

  • Use CHDH antibodies to track mitochondrial changes following neurotoxic insults

  • Combine with live-cell imaging to correlate CHDH dynamics with functional outcomes

• Mechanistic investigation approaches:

  • Examine CHDH-protein interactions in brain tissue using co-immunoprecipitation

  • Assess post-translational modifications of CHDH in disease states

  • Correlate CHDH expression with choline metabolite levels in affected tissues

• Therapeutic intervention monitoring:

  • Use CHDH immunoassays to track responses to metabolic interventions

  • Monitor CHDH expression changes following neuroprotective treatments

  • Develop CHDH activity assays following immunocapture from brain tissue

• Human biomarker development:

  • Explore CHDH detection in exosomes or circulating mitochondria as potential biomarkers

  • Correlate CSF or plasma CHDH levels with disease progression

  • Develop ultrasensitive immunoassays for early detection of metabolic dysregulation

These methodological approaches leverage CHDH antibodies to explore the intersection of choline metabolism, mitochondrial function, and neurodegeneration, potentially revealing novel therapeutic targets or biomarkers .