LDHA Antibody

What is LDHA and why is it significant in research?

LDHA (Lactate Dehydrogenase A) is a metabolic enzyme that preferentially catalyzes the conversion of pyruvate to lactate. It plays a crucial role in energy metabolism and protein transport processes, with potential implications in muscle development. The A subunits of LDH predominantly appear in skeletal muscle, while B subunits are abundantly produced in brain and heart. LDHA has been identified as a "disallowed gene" in pancreatic β-cells, meaning its expression is normally suppressed in these cells but becomes upregulated under diabetic conditions. This makes it a valuable marker for metabolic dysregulation research and a potential therapeutic target .

What are the primary applications for LDHA antibodies in research?

LDHA antibodies can be employed in multiple experimental applications:

• Western Blot (WB): Typically used at 1:5000-1:30000 dilution for detecting LDHA protein (32-37 kDa) in cell and tissue lysates

• Immunohistochemistry (IHC): Used at 1:20-1:200 dilution for detecting LDHA in fixed tissue sections

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Used at 1:50-1:500 dilution for cellular localization studies

• Immunoprecipitation (IP): Typically 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

• ELISA: For quantitative detection of LDHA in solution

What tissues and cell types show positive reactivity with LDHA antibodies?

LDHA antibodies have demonstrated positive reactivity in multiple tissues and cell types:

This broad reactivity across species makes these antibodies valuable for comparative studies across human, mouse, and rat experimental models .

What are the molecular characteristics of LDHA important for antibody selection?

When selecting an LDHA antibody, consider these molecular characteristics:

• Calculated molecular weight: 332 amino acids, approximately 37 kDa

• Observed molecular weight on gels: 32-37 kDa (may appear at 34-36 kDa in some systems)

• Some detection systems may show bands at approximately 38 kDa

• Gene ID (NCBI): 3939

• UniProt ID: P00338

• GenBank Accession Number: BC067223

• LDHA can form homotetramers and has multiple isoforms ranging from 27-40 kDa

These details are essential for properly interpreting Western blot results and choosing antibodies with appropriate epitopes for your experimental system .

How should LDHA antibodies be validated for specificity in experimental systems?

Comprehensive validation of LDHA antibodies should include:

• Knockout/Knockdown Controls: Use LDHA knockout or knockdown cell lines to confirm antibody specificity. Multiple publications have used this approach for validating anti-LDHA antibodies.

• Peptide Competition Assays: For post-translational modification-specific antibodies (like succinyl-K118), perform competitive immunoblots using synthesized peptides. For example, the specificity of succinyl-LDHA K118 antibody can be confirmed by dot-blot assay and competitive immunoblot using synthesized LDHA succinyl-K118 peptide .

• Cross-Reactivity Assessment: Test the antibody across multiple species if your research involves comparative studies. Current antibodies show reactivity with human, mouse, rat, and sometimes pig and monkey samples .

• Multiple Detection Methods: Validate using orthogonal techniques (WB, IP, IHC, IF) to ensure consistent results across platforms. Discrepancies between methods may indicate non-specific binding in certain applications .

For Western Blot:

• Use RIPA or Immunoblot Buffer Group 1 for optimal extraction

• Include protease inhibitors to prevent degradation

• Run gels under reducing conditions

• Load 0.5 mg/ml of lysate for consistent detection

• Use PVDF membrane for optimal protein transfer

For Immunohistochemistry:

• For paraffin-embedded tissues: Use TE buffer (pH 9.0) for antigen retrieval, though citrate buffer (pH 6.0) may be used as an alternative

• For optimal staining, use 3 μg/mL antibody concentration and incubate for 1 hour at room temperature

• Use DAB (brown) for visualization and counterstain with hematoxylin (blue)

• Specific staining for LDHA is typically localized to cytoplasm in most cells

For Immunofluorescence:

• Fix cells with 4% paraformaldehyde

• Permeabilize with 0.1% Triton X-100

• Block with 1-5% BSA

• Incubate with primary antibody (3 μg/mL) for 3 hours at room temperature

• Use fluorophore-conjugated secondary antibodies (like NorthernLights 557) for detection

• Counterstain nuclei with DAPI

• Look for cytoplasmic localization of LDHA

How can researchers distinguish between LDHA and other LDH isoforms?

Distinguishing between LDH isoforms requires:

• Isoform-Specific Epitopes: Select antibodies targeting unique regions of LDHA not shared with LDHB or other isoforms. Antibodies raised against the Ala2-Val92 region of human LDHA can provide specificity .

• Expression Pattern Analysis: LDHA predominates in skeletal muscle while LDHB is more abundant in brain and heart. Tissue-specific expression patterns can help confirm isoform identity .

• Molecular Weight Differentiation: Although subtle, LDHA and LDHB may migrate slightly differently on gels (LDHA: 32-37 kDa). Use high-resolution gels for separation .

• Activity Assays: LDHA preferentially catalyzes pyruvate to lactate conversion, while LDHB favors the reverse reaction. Combining antibody detection with activity assays provides functional confirmation .

• Knockout Controls: Use cells with specific isoform knockouts to confirm antibody specificity and rule out cross-reactivity .

How can LDHA antibodies be used to study metabolic reprogramming in cancer?

LDHA antibodies are valuable tools for investigating the Warburg effect and metabolic reprogramming in cancer:

• Expression Level Analysis: Use Western blot with LDHA antibodies to quantify upregulation in cancer cell lines (like Daudi, HepG2, MOLT-4) compared to normal tissues. Multiple studies have shown increased LDHA expression correlates with cancer progression .

• Tissue Distribution Studies: Employ IHC to map LDHA expression patterns across tumor sections, identifying metabolically distinct regions. This reveals heterogeneity in glycolytic metabolism within tumors .

• Subcellular Localization: Use immunofluorescence to track potential changes in LDHA localization in response to hypoxia or other metabolic stressors. LDHA typically shows cytoplasmic localization but may form complexes with other metabolic enzymes under stress conditions .

• Post-Translational Modification Analysis: Specialized antibodies detecting modified LDHA (such as succinylated K118) can reveal regulatory mechanisms. Succinylation at K118 has been shown to increase LDHA activity and enhance cancer cell invasion capabilities .

• Therapeutic Response Monitoring: Track changes in LDHA expression following treatment with metabolism-targeting therapeutics to assess efficacy and mechanism of action .

What protocols are recommended for studying LDHA in pancreatic islet research?

For investigating LDHA in pancreatic islet biology, particularly in diabetes research:

• Cell-Type Specific Detection: Use co-immunostaining with cell-type markers (insulin for β-cells, glucagon for α-cells) alongside LDHA antibodies at 1:20-1:200 dilution to determine cell-specific expression patterns. Current research shows LDHA is mainly localized in human α-cells while expressed at very low levels in β-cells under normal conditions .

• Diabetic Model Analysis: Compare LDHA expression in islets from normal vs. diabetic sources using Western blot and IHC. Evidence shows LDHA is upregulated in pancreatic islets exposed to chronic high glucose treatment and in autopsy pancreases from individuals with type 2 diabetes .

• Functional Studies: Combine LDHA detection with measurements of insulin secretion and glucagon release to correlate expression with functional outcomes. Pharmacological inhibition of LDHA in isolated human islets has been shown to enhance insulin secretion under physiological conditions .

• Lactate Production Assay: Pair LDHA protein detection with functional lactate production assays to connect expression changes with metabolic outcomes. Protocols using 0.1g tissues or 1×10^6 cells homogenized in cold assay buffer can measure LDHA activity via colorimetric assays .

• Single-Cell Analysis: Integrate LDHA antibody staining with single-cell RNA-seq data to correlate protein expression with transcriptional profiles across islet cell populations .

What considerations are important when interpreting LDHA antibody signals across different experimental systems?

When interpreting LDHA antibody results, researchers should consider:

What are common issues encountered with LDHA antibodies and how can they be resolved?

How can researchers develop and validate custom antibodies against modified forms of LDHA?

For developing specialized antibodies against modified LDHA (e.g., succinylated, phosphorylated):

• Peptide Design: Select peptides containing the modification of interest in the context of surrounding amino acids. For succinyl-K118 LDHA antibodies, peptides like CRNVNIF-Ksu-FIIPNVVK and RKRNVNIF-Ksu-FIIPNC have been successfully used .

• Carrier Protein Conjugation: Conjugate the modified peptide to a carrier protein like KLH (Keyhole Limpet Hemocyanin) for immunization .

• Host Selection: Choose rabbits for polyclonal antibody generation or consider mouse/rat for monoclonal antibody development .

• Antibody Purification Strategy: Implement a two-step purification process:

  • First deplete antisera with unmodified peptides to remove antibodies recognizing the unmodified form

  • Then perform affinity purification using the modified peptide to isolate modification-specific antibodies

• Validation Experiments:

  • Dot-blot assays comparing modified vs. unmodified peptides

  • Competitive immunoblotting with excess free peptide

  • Testing on samples with known modification status (e.g., cells with SIRT5 knockout for succinylation studies)

  • Parallel validation with mass spectrometry data

What are the optimal storage conditions for maintaining LDHA antibody performance?

To ensure long-term stability and performance of LDHA antibodies:

• Storage Temperature: Store at -20°C for long-term stability. Refrigeration at 4°C is only suitable for short-term storage (typically less than two weeks) .

• Buffer Composition: Optimal storage buffers typically contain:

  • PBS base with pH 7.3

  • 50% glycerol as a cryoprotectant

  • 0.02% sodium azide as a preservative

  • Some formulations include 0.5% BSA for additional stability

• Aliquoting Recommendations: For smaller sizes (≤20μl), aliquoting is generally unnecessary for -20°C storage. For larger volumes, create single-use aliquots to minimize freeze-thaw cycles .

• Special Considerations for Conjugated Antibodies: For fluorophore-conjugated antibodies (like CoraLite Plus 488-conjugated LDHA antibodies):

  • Protect from light exposure during storage and handling

  • Store in buffer containing 0.05% Proclin300 instead of sodium azide

  • Maintain the same -20°C storage temperature

• Shelf-Life: When properly stored, antibodies typically remain stable for one year after shipment, though actual performance may extend beyond this period with proper handling .

How can LDHA antibodies contribute to research on LDHA as a cancer metastasis biomarker?

LDHA antibodies are increasingly valuable in cancer metastasis research:

• Post-Translational Modification Studies: Using specialized antibodies against succinylated LDHA at K118 can help identify regulatory mechanisms. Research has shown that succinylation at K118 increases LDHA activity and enhances cancer cell invasion capabilities, positioning it as a potential metastatic biomarker .

• Comparative Expression Analysis: LDHA antibodies enable quantitative comparison of LDHA levels between primary tumors and metastatic lesions using IHC and Western blot. Multiple studies have correlated elevated LDHA with increased metastatic potential .

• Functional Assessment: Combining expression data from antibody-based detection with LDHA activity assays provides a more comprehensive picture of metabolic changes during metastasis. Activity assays using 0.1g tissues or 1×10^6 cells homogenized in cold assay buffer can be measured via colorimetric methods .

• Therapeutic Target Validation: LDHA antibodies help validate the efficacy of LDHA-targeting therapeutic approaches by confirming target engagement and expression reduction in pre-clinical models. This is particularly relevant as LDHA inhibitors are being developed as potential anti-metastatic agents .

• Multi-marker Panels: Integrating LDHA detection with other metastasis-associated proteins creates more robust biomarker panels. Co-immunostaining approaches can reveal relationships between LDHA and other metabolic enzymes or signaling proteins involved in metastasis .

What role does LDHA play in pancreatic islet function and how can antibodies help study this?

LDHA antibodies reveal important insights into pancreatic islet metabolism and diabetes:

• Cell-Type Specific Expression Patterns: LDHA antibodies combined with cell markers demonstrate that LDHA is primarily expressed in α-cells while nearly absent in β-cells under normal conditions. This cell-specific distribution suggests distinct metabolic programming between islet cell types .

• Pathological Upregulation: In diabetic conditions, LDHA antibodies reveal inappropriate upregulation in islet cells. Both chronic high glucose exposure and type 2 diabetes are associated with increased LDHA expression, primarily in α-cells but potentially also in β-cells .

• Metabolic Consequences: By correlating LDHA expression with functional assays, researchers have shown that pharmacological inhibition of LDHA in isolated human islets enhances insulin secretion under physiological conditions. This suggests LDHA expression may contribute to β-cell dysfunction in diabetes .

• "Disallowed Gene" Concept Validation: LDHA is considered a "disallowed gene" in β-cells, meaning its expression is normally suppressed. Antibody-based detection confirms this concept in human islets and reveals how this suppression is compromised in pathological states .

• Species Differences: Unlike rodent models where LDHA re-expression in β-cells is well-documented in diabetes, human studies using LDHA antibodies show upregulation primarily in α-cells. This highlights important species differences in metabolic adaptation to diabetes .