LDHC Antibody

What is LDHC and why is it significant in scientific research?

LDHC, also known as LDH-X and CT32, belongs to the lactate dehydrogenase family. It plays critical roles in glycolysis and energy supply, particularly in male germ cells. LDHC is significant in research for several reasons:

• It was one of the first testis-specific isozymes discovered in male germ cells

• It's essential for sperm motility and male fertility

• It has emerging roles in cancer biology as a cancer testis antigen

• Its tissue-specific expression pattern makes it an excellent model for studying gene regulation

LDHC differs from the ubiquitously expressed LDHA and LDHB isozymes in its restricted expression pattern and functional properties. While heterotetramers containing both A and C subunits are not detected in murine or human testes, LDHC remains the predominant LDH in germ cells .

What are the typical expression patterns of LDHC in normal and pathological tissues?

Normal tissues:

• Primarily expressed in testis (specifically in preleptotene spermatocytes, spermatids, and spermatozoa)

• Detected in the principal piece of spermatozoa with weaker signal in the midpiece region

• Historically thought to be exclusively testis-specific, but newer research suggests potential expression in other tissues under specific conditions

Pathological tissues:

• Aberrantly expressed in multiple cancer types, particularly:

  • Breast cancer (especially basal-like and HER2-enriched subtypes)

  • Renal cell carcinoma (correlated with shorter progression-free survival)

How do I select the appropriate LDHC antibody for my specific application?

Selection of an appropriate LDHC antibody depends on several critical factors:

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, IHC, IF, IP). For example, antibody 19989-1-AP has been validated for WB, IHC, IF/ICC, IP, and ELISA applications .

• Species reactivity: Confirm reactivity with your species of interest. Some LDHC antibodies show cross-reactivity across human, mouse, and rat samples .

• Epitope consideration: For localization studies, consider antibodies targeting different epitopes. Studies have used peptide-specific antibodies such as MC5-15 and MC211-220 to detect LDHC in the principal piece of spermatozoa .

• Validation evidence: Review published literature citing the antibody and examine validation data provided by manufacturers. For antibody 19989-1-AP, validation data includes positive detection in multiple tissues and cell lines .

• Isotype and host: Consider the host species (e.g., rabbit IgG) to ensure compatibility with your experimental design, particularly for co-staining experiments .

What are effective methods for validating LDHC antibody specificity?

Validating LDHC antibody specificity is crucial for reliable experimental results. Consider these methodological approaches:

• Knockout/knockdown controls:

  • Use tissues/cells from Ldhc knockout mice as negative controls

  • Compare signal between LDHC-silenced cancer cells and their control counterparts

  • Utilize human LDHC knock-in (hLDHC KI) mice for comparison with wild-type mice

• Peptide competition assays:

  • Pre-incubate the antibody with the immunizing peptide

  • Compare staining patterns with and without peptide competition

• Multiple antibody validation:

  • Compare staining patterns using antibodies targeting different LDHC epitopes

  • Verify consistent molecular weight detection (30-36 kDa range)

• Tissue panel analysis:

  • Test antibody across multiple tissues with known LDHC expression profiles

  • Include testis tissue as a positive control and non-reproductive tissues as negative controls

• Recombinant protein controls:

  • Use purified recombinant LDHC protein as a positive control

  • Test cross-reactivity with purified LDHA and LDHB proteins to ensure specificity

What are the optimal conditions for detecting LDHC by Western blot?

Detecting LDHC via Western blot requires specific optimization strategies:

Sample preparation:

• For testicular tissue: homogenize in RIPA buffer with protease inhibitors

• For spermatozoa: special attention to membrane solubilization may be needed

• For cancer cells: standard cell lysis protocols are typically sufficient

Protein loading and transfer:

• Load 10-20 μg of total protein per lane

• Use PVDF membrane for better protein retention

• Transfer at lower voltage (e.g., 30V overnight at 4°C) for improved transfer efficiency

Antibody dilution and incubation:

• Primary antibody: 1:200-1:1000 dilution range (antibody-dependent)

• Example protocol: block with 5% milk for 1 hour at room temperature, then incubate with primary antibody overnight at 4°C

• Secondary antibody: typically 1:5000 dilution of HRP-conjugated antibody appropriate for host species

Expected results:

• LDHC typically appears at 30-36 kDa

• In some tissues, additional bands may be present due to splice variants or post-translational modifications

Troubleshooting:

• High background: increase blocking time or stringency of wash steps

• Weak signal: increase antibody concentration or extend incubation time

• Multiple bands: validate specificity with knockout controls

How should immunohistochemistry protocols be optimized for LDHC detection?

Successful LDHC immunohistochemistry requires careful protocol optimization:

Tissue preparation:

• Fix tissues in 4% paraformaldehyde or 10% neutral buffered formalin

• For testicular tissue, careful fixation timing is critical to preserve antigenicity while allowing proper penetration

Antigen retrieval:

• Recommended: TE buffer at pH 9.0

• Alternative: citrate buffer at pH 6.0

• Heat-induced epitope retrieval (HIER) is typically more effective than enzymatic methods

Antibody dilution:

• Recommended range: 1:200-1:800 for IHC applications

• Titrate antibody with positive control tissue (testis) to determine optimal concentration

Detection system:

• Both DAB and fluorescence-based detection systems are suitable

• For low expression tissues, consider signal amplification methods (e.g., tyramide signal amplification)

Controls:

• Positive control: human or mouse testis tissue

• Negative controls: include primary antibody omission and non-reproductive tissues

• Consider using tissues from Ldhc knockout mice as specificity controls

Expected results:

• Strong staining in testicular germ cells, particularly spermatocytes and spermatids

• In cancer tissues, staining patterns may be heterogeneous

How can LDHC antibodies be utilized to investigate male fertility issues?

LDHC antibodies offer powerful tools for male fertility research:

Sperm function analysis:

• Immunolocalization of LDHC in human and animal spermatozoa reveals its distribution primarily in the principal piece with weaker signal in the midpiece region

• Changes in LDHC localization or abundance may correlate with impaired sperm motility

Methodology for sperm analysis:

• Collect and wash sperm samples in PBS

• Fix with 4% paraformaldehyde

• Permeabilize with 0.1% Triton X-100

• Block with appropriate serum

• Incubate with LDHC antibody (1:10-1:100 dilution for IF)

• Visualize with fluorescent secondary antibody

• Counterstain nucleus with DAPI

Knockout models:

• Ldhc knockout mice exhibit impaired sperm motility, demonstrating the critical role of LDHC in sperm function

• Human LDHC knock-in mice (hLDHC KI) can serve as models to assess human LDHC-targeting contraceptive drugs

Clinical applications:

• LDHC antibodies can potentially detect abnormalities in LDHC expression or localization in sperm from infertile men

• Correlation of LDHC antibody staining patterns with computer-assisted sperm analysis (CASA) parameters may provide insights into functional defects

What role does LDHC play in cancer research and how can LDHC antibodies contribute?

LDHC has emerging roles in cancer biology with significant research applications:

Cancer expression profiling:

• LDHC is aberrantly expressed in multiple cancer types, particularly in basal-like and HER2-enriched breast cancers

• LDHC expression correlates with poor survival, larger tumor size, and recurrence in breast cancer patients

Mechanistic studies:

• Targeting LDHC in breast cancer cells dysregulates the cell cycle and increases DNA damage, compromising long-term cell survival

• LDHC knockdown experiments reveal effects on cancer cell proliferation, migration, and invasion

Immunomodulatory effects:

• LDHC silencing in cancer cells enhances T cell activation and cytolytic activity

• LDHC knockdown increases tumor-derived GM-CSF, IFN-γ, MCP-1, and CXCL1 while decreasing IL-6 and Gal-9 production

• LDHC expression in melanoma and breast tumors is associated with T cell dysfunction and potentially affects immunotherapy responses

Experimental approaches using LDHC antibodies:

• Western blot analysis to quantify LDHC expression levels in cancer tissues and cell lines

• Immunohistochemistry to assess LDHC distribution in tumor tissues

• Immunofluorescence to study LDHC subcellular localization in cancer cells

• Immunoprecipitation to identify LDHC-interacting proteins in tumor contexts

How can we resolve discrepancies in LDHC antibody detection across different experimental systems?

Discrepancies in LDHC antibody detection can arise from multiple factors:

Source of variation and resolution strategies:

• Antibody epitope specificity:

  • Different antibodies may recognize distinct epitopes that are differentially accessible in various experimental conditions

  • Solution: Compare multiple antibodies targeting different LDHC epitopes

• Splice variants:

  • Some studies report tumor-specific LDHC isoforms with defects in the catalytic domain

  • Solution: Design primers/antibodies targeting common and variant-specific regions

• Cross-reactivity with LDHA/LDHB:

  • LDHC shares sequence homology with other LDH family members

  • Solution: Validate specificity against purified LDHA and LDHB proteins, and include Ldhc knockout controls

• Post-translational modifications:

  • PTMs may mask epitopes in certain contexts

  • Solution: Try multiple antibodies and antigen retrieval methods; consider phosphospecific antibodies if relevant

• Subcellular localization variability:

  • LDHC may have different accessibility in different subcellular compartments

  • Reports of LDHC in sperm mitochondrial matrix and cortical regions require specific extraction methods

  • Solution: Optimize fixation and permeabilization protocols for each application

Methodological approach to resolve discrepancies:

• Perform parallel experiments with multiple validated antibodies

• Include appropriate positive and negative controls (tissue, genetic)

• Complement antibody-based methods with molecular techniques (qPCR, mass spectrometry)

• Document and report all experimental conditions precisely

How are LDHC antibodies being used to develop male contraceptive approaches?

LDHC represents a promising target for male contraceptive development:

Research approaches using LDHC antibodies:

• Humanized mouse models: Human LDHC knock-in (hLDHC KI) mice serve as valuable tools to assess LDHC-targeting contraceptive drugs in preclinical studies

• Drug screening methodologies:

  • Computer-assisted sperm analysis (CASA) coupled with LDHC antibody validation

  • In vitro fertilization (IVF) assessments before and after treatment with LDH inhibitors

  • Comparative analysis between wild-type and hLDHC KI mice to evaluate human-specific effects

• Mode of action studies:

  • Immunolocalization of LDHC before and after drug treatment

  • Assessment of phosphorylation status and other post-translational modifications

  • Correlation of LDHC inhibition with functional parameters of sperm motility

Key experimental findings:

• LDH inhibitors more specific to human LDHC than mouse LDHC reduce fertilization rates in hLDHC KI mice but not in wild-type mice

• This species specificity highlights the importance of humanized models for contraceptive development

• LDHC's role in glycolysis and ATP production makes it a rational target for inhibiting sperm motility without affecting other tissues

What are the latest techniques for studying LDHC interaction with other proteins in cellular contexts?

Advanced techniques for studying LDHC protein interactions include:

Immunoprecipitation-based approaches:

• Co-immunoprecipitation using LDHC antibodies (recommended: 0.5-4.0 μg antibody for 1.0-3.0 mg total protein lysate)

• Proximity ligation assay (PLA) to detect and visualize LDHC interactions with other proteins in situ

• Mass spectrometry analysis of LDHC immunoprecipitates to identify novel binding partners

Live-cell imaging techniques:

• Fluorescent protein tagging combined with LDHC antibody validation

• FRET/BRET analysis to study dynamic interactions in living cells

• Super-resolution microscopy with LDHC antibodies to examine nanoscale protein complexes

Cross-linking strategies:

• In vivo cross-linking followed by LDHC immunoprecipitation

• BioID or APEX2 proximity labeling with LDHC fusion proteins

• Validation of identified interactions using reciprocal co-immunoprecipitation

Functional interaction studies:

• Co-localization of LDHC with other glycolytic enzymes in sperm flagella

• Investigation of LDHC interaction with mitochondrial proteins in sperm midpiece

• Analysis of LDHC associations with cytoskeletal elements that may affect sperm motility

When designing these experiments, researchers should carefully validate LDHC antibodies for specificity in the selected application and consider epitope accessibility in protein complexes.

What are common challenges in LDHC detection and how can they be overcome?

Researchers face several challenges when detecting LDHC:

Challenge 1: Low signal-to-noise ratio

• Causes: Insufficient antibody concentration, inadequate antigen retrieval, low LDHC expression

• Solutions:

  • Optimize antibody dilution (test range: 1:200-1:1000 for WB, 1:200-1:800 for IHC)

  • Try different antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

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

  • Use signal amplification systems for low-expression samples

Challenge 2: Non-specific binding

• Causes: Cross-reactivity with other LDH isozymes, insufficient blocking

• Solutions:

  • Increase blocking time and concentration (e.g., 5% milk for 1 hour)

  • Use additional blocking agents (e.g., normal serum matching secondary antibody species)

  • Increase wash stringency and duration

  • Validate results with Ldhc knockout negative controls

Challenge 3: Inconsistent results across different sample types

• Causes: Variable fixation effects, tissue-specific processing requirements

• Solutions:

  • Standardize fixation protocols for each tissue type

  • Optimize permeabilization conditions for different cell types

  • Consider membranous localization of LDHC in some contexts

  • Use parallel detection methods (WB, IHC, IF) for confirmation

Challenge 4: Unexpected molecular weight bands

• Causes: Splice variants, degradation products, post-translational modifications

• Solutions:

  • Use freshly prepared samples with protease inhibitors

  • Run gradient gels to better resolve proteins in the 30-36 kDa range

  • Include positive controls (testis tissue) to benchmark expected band size

  • Consider phosphorylation status effects on migration

How can researchers interpret conflicting LDHC antibody results in cancer studies?

Cancer research with LDHC antibodies requires careful result interpretation:

Sources of conflicting results:

• Tumor heterogeneity:

  • LDHC expression may vary within different regions of the same tumor

  • Solution: Use multiple sampling within tumors and document specific regions analyzed

• Cancer subtype differences:

  • LDHC expression varies across breast cancer subtypes (higher in basal-like and HER2-enriched)

  • Solution: Stratify analysis by molecular subtype and compare within defined groups

• Methodological variations:

  • Different antibodies and detection methods yield varying sensitivity

  • Solution: Standardize protocols and use multiple antibodies targeting different LDHC epitopes

• Splice variant detection:

  • Tumor-specific LDHC isoforms with defects in catalytic domains may exist

  • Solution: Use antibodies recognizing different domains and complement with RT-PCR analysis

Analytical approach to resolve conflicts:

• Multimodal validation:

  • Combine protein detection (antibody-based) with mRNA analysis

  • Verify functional activity using enzymatic assays

  • Correlate LDHC expression with clinical parameters across larger cohorts

• Comparative analysis:

  • Use consistent methodologies across different tumor types

  • Compare cancer tissues with matched normal tissues from the same patients

  • Include testicular tissue as positive control in all experiments

• Functional knockdown studies:

  • Validate antibody specificity using LDHC-silenced cancer cells

  • Correlate LDHC protein levels with phenotypic effects following knockdown

  • Analyze downstream effects on cell cycle, DNA damage, and immune responses