LDHA Antibody Pair

What is LDHA and why is it an important target for antibody-based detection in research?

Lactate dehydrogenase A (LDHA) is an enzyme composed of A subunits that predominantly occurs in skeletal muscle, while B subunits are abundantly produced in brain and heart. LDHA plays a crucial role in energy metabolism, catalyzing the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD(+) . The significance of LDHA in research stems from its involvement in key metabolic processes and its altered expression in various disease states.

LDHA exists as multiple isoforms with molecular weights ranging from 27-40 kDa and can form homotetramers. The protein has a calculated molecular weight of approximately 37 kDa, consisting of 332 amino acids, though its observed molecular weight in experimental conditions typically ranges between 32-40 kDa . LDHA's role in cancer metabolism (Warburg effect) and connection to cellular proliferation makes it a valuable target for oncology research, particularly in investigating metabolic reprogramming in cancer cells .

What types of LDHA antibodies are available for research applications and how do they differ?

Based on the search results, several types of LDHA antibodies are available for research use:

For instance, product CL488-21799 is a polyclonal rabbit antibody conjugated to CoraLite® Plus 488 Fluorescent Dye, optimized for immunofluorescence applications . In contrast, M00825 is a rabbit monoclonal antibody suitable for multiple applications including WB, IHC, ICC/IF, IP, and Flow Cytometry . The choice between these antibody types depends on the specific research requirements, desired sensitivity, and application.

What are the recommended dilution ratios and experimental conditions for LDHA antibodies across different applications?

The optimal dilution ratios for LDHA antibodies vary based on the specific application and antibody product:

For Immunofluorescence (IF)/ICC:

• Polyclonal antibodies (e.g., CL488-21799): 1:50-1:500

• Monoclonal antibodies (e.g., GTX101416): 1:1000

For Western Blot (WB):

• Polyclonal antibodies: 1:500-1:3000

• Monoclonal antibodies: 1:1000 (typical)

For Immunohistochemistry (IHC):

• 1:100-1:400

For Flow Cytometry (Intracellular):

• 0.25 μg per 10^6 cells in a 100 μl suspension

For Sandwich ELISA:

• Capture antibody (e.g., ab317640): 2 μg/mL

• Detector antibody (e.g., ab317641): 0.5 μg/mL

It's recommended to titrate each antibody in the specific testing system to obtain optimal results, as performance can be sample-dependent . For Western blot, experimental conditions often involve using 10% SDS-PAGE gels, with proteins transferred to nitrocellulose or PVDF membranes, followed by blocking with 5% non-fat milk/TBS .

How should LDHA antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of LDHA antibodies is critical to maintain their activity and specificity:

Storage Conditions:

• Store at -20°C for long-term stability (typical recommendation)

• For conjugated antibodies: Avoid exposure to light

• For short-term storage and frequent use: 4°C for up to one month (for some products)

• Avoid repeated freeze-thaw cycles

Storage Buffer Components:

• PBS with 50% Glycerol, 0.05% Proclin300, 0.5% BSA, pH 7.3 (for some products)

• Some products contain sodium azide (0.02%) as a preservative

How can researchers validate the specificity of LDHA antibodies in their experimental systems?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For LDHA antibodies, several validation strategies are recommended:

1. Western Blot Validation:

• Confirm the detection of a single band at the expected molecular weight (32-40 kDa for LDHA)

• Use positive control samples known to express LDHA (e.g., HepG2, A431, Daudi cells, or skeletal muscle tissue)

• Include negative controls where possible

2. Knockout/Knockdown Validation:

• Use LDHA knockout or knockdown samples as negative controls

• Search results mention CRISPR/Cas9 gene editing and LDHA-specific siRNA as tools for creating validation controls

3. Multiple Antibody Validation:

• Compare results using antibodies targeting different epitopes of LDHA

• Use both monoclonal and polyclonal antibodies to confirm specificity

4. Cross-Reactivity Testing:

• Verify species reactivity as claimed by manufacturers

• Test antibodies across relevant research models (human, mouse, rat samples)

5. Immunoprecipitation Followed by Mass Spectrometry:

• For definitive validation, immunoprecipitate LDHA and confirm identity by mass spectrometry

Researchers should include appropriate experimental controls and validate each antibody in their specific experimental system before proceeding with critical experiments.

What are the recommended positive control samples for LDHA antibody testing?

Based on the search results, several cell lines and tissue types are recommended as positive controls for LDHA antibody testing:

Cell Lines:

• HepG2 cells: Positive for IF/ICC applications

• A431 cells: Positive for Western blot

• HeLa cells: Suitable for immunofluorescence

• MCF7 cells: Used for immunofluorescence validation

• Daudi, MOLT-4, and HepG2 cell lines: Validated for Western blot

Tissue Samples:

• Rat skeletal muscle: Positive for Western blot

• Mouse skeletal muscle: Positive for Western blot

• Pig skeletal muscle and liver tissues: Positive for Western blot

• Human liver cancer tissue: Positive for IHC

These positive controls provide reliable reference points for validating antibody performance across different applications. For robust validation, researchers should ideally include multiple positive controls representing different tissue or cell types relevant to their specific research focus.

What are the important considerations for species reactivity when selecting LDHA antibodies?

Species reactivity is a critical consideration when selecting LDHA antibodies for comparative or translational studies. From the search results, we can extract the following guidance:

Common Species Reactivity Patterns:

• Many LDHA antibodies react with human, mouse, and rat samples

• Some antibodies additionally react with pig samples

• Zebrafish reactivity is mentioned for certain recombinant antibodies

Cross-Reactivity Considerations:

• Despite high sequence homology between species, not all antibodies will cross-react equally

• Some antibodies may show stronger reactivity with certain species over others

• Epitope accessibility may vary between species due to subtle structural differences

Validation Across Species:

• Researchers should verify cross-reactivity claims with experimental validation

• Different applications may show different cross-reactivity profiles for the same antibody

• Control samples from each relevant species should be tested

Application-Specific Considerations:

• For Western blot, cross-reactivity is often more reliable due to denatured protein state

• For applications requiring native protein recognition (IF, IHC), species-specific validation is more critical

When working with animal models, selecting an antibody with validated reactivity across your species of interest is essential for comparative studies and translational research.

How can LDHA antibody pairs be optimized for sandwich ELISA applications?

Sandwich ELISA using LDHA antibody pairs requires careful optimization for maximal sensitivity and specificity. Based on the search results, here are key considerations:

Antibody Pair Selection:

• Use antibodies targeting different, non-overlapping epitopes of LDHA

• The Abcam antibody pair (ab317639) consists of:

  • Capture antibody [EPR25915-6] (ab317640) used at 2 μg/mL

  • Detector antibody [EPR25915-88] (ab317641) used at 0.5 μg/mL

Optimization Steps:

• Antibody Concentration Titration:

  • Test a range of concentrations for both capture and detector antibodies

  • Typical starting points: 1-5 μg/mL for capture and 0.1-1 μg/mL for detector antibodies

• Buffer Optimization:

  • Test different coating buffers (carbonate/bicarbonate pH 9.6, PBS pH 7.4)

  • Optimize blocking buffers (BSA vs. non-fat milk)

  • Evaluate sample diluents to minimize matrix effects

• Incubation Conditions:

  • Optimize temperature (4°C, room temperature, 37°C)

  • Determine optimal incubation times for each step

• Standard Curve Development:

  • Use recombinant LDHA protein for standard curve generation

  • Establish the linear range and lower limit of detection

• Validation with Known Samples:

  • Test samples with known LDHA concentrations

  • Perform spike-and-recovery experiments

  • Assess intra- and inter-assay variability

The performance of the antibody pair will depend on the specific characteristics of the assay and sample type . Researchers should note that carrier-free formulations like those mentioned in the search results (BSA and azide free) are particularly suitable for sandwich ELISA applications as they minimize background and increase specificity .

What are the methodological approaches for investigating LDHA in cancer metabolism research?

LDHA plays a pivotal role in cancer metabolism, particularly in the Warburg effect. Advanced research approaches using LDHA antibodies include:

1. Expression Analysis in Cancer vs. Normal Tissues:

• Immunohistochemistry using LDHA antibodies can reveal differential expression

• Example protocol:

  • Antigen retrieval with EDTA buffer (pH 8.0) or citrate buffer (pH 6.0)

  • Blocking with 10% goat serum

  • LDHA antibody incubation (1:200 dilution) overnight at 4°C

  • Detection with appropriate secondary antibody and DAB chromogen

2. Metabolic Phenotyping via LDHA Inhibition:

• Combine LDHA antibody detection with metabolic inhibitors (e.g., oxamate)

• Monitor changes in cell viability, colony formation, and apoptosis

3. Signaling Pathway Analysis:

• Investigate relationships between LDHA and oncogenic pathways

• The search results mention connections between HER2 and LDHA in gastric cancer

• Western blot protocols for multiple protein detection:

  • Separate proteins on 5-20% SDS-PAGE

  • Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

  • Block with 5% non-fat milk/TBS

  • Incubate with anti-LDHA antibody (1:500-1:3000) overnight at 4°C

  • Develop using enhanced chemiluminescence

4. Genetic Manipulation Approaches:

• Use CRISPR/Cas9 or siRNA to knockdown LDHA

• The search results describe LDHA-specific siRNA (sc-43893) for knockdown studies

• CRISPR/Cas9 targeting of LDHA with the guide sequence 5′-TAGGAGGCTATGGACTTGCAGCA-3′

• Antibodies can validate knockdown efficiency and monitor phenotypic effects

5. ROS Production Analysis:

• Combine LDHA antibody detection with ROS assays

• Investigate the relationship between LDHA activity and oxidative stress in cancer cells

These methodological approaches provide a framework for comprehensive investigation of LDHA's role in cancer metabolism, enabling researchers to explore therapeutic targeting strategies.

How can researchers optimize immunofluorescence protocols for LDHA detection in various cell types?

Optimizing immunofluorescence (IF) protocols for LDHA detection requires attention to several critical factors:

Fixation and Permeabilization:

• Most protocols use 4% paraformaldehyde fixation at room temperature for 15 minutes

• Permeabilization with 0.1-0.5% Triton X-100 is commonly used for intracellular targets like LDHA

Antibody Selection and Dilution:

• Fluorescently conjugated antibodies (e.g., CL488-21799) simplify the protocol by eliminating secondary antibody steps

• Dilution ranges for LDHA antibodies in IF:

  • Polyclonal antibodies: 1:50-1:500

  • Monoclonal antibodies: Often used at higher dilutions (1:1000)

Counterstaining and Controls:

• Incorporate counterstains for reference structures:

  • Phalloidin for cytoskeleton (1:200)

  • Hoechst 33342 for nuclear staining

• Include positive controls (HepG2, HeLa, or MCF7 cells)

• Negative controls should omit primary antibody

Advanced Protocol for Maximum Sensitivity:

• Grow cells on coated coverslips to 70-80% confluence

• Fix with 4% paraformaldehyde (15 min, RT)

• Wash 3× with PBS

• Permeabilize with 0.2% Triton X-100 (10 min, RT)

• Block with 5% normal serum in PBS (1 hour, RT)

• Incubate with LDHA antibody at optimized dilution (overnight, 4°C)

• Wash 3× with PBS

• For unconjugated antibodies: Incubate with fluorophore-conjugated secondary antibody (1 hour, RT)

• Counterstain as needed

• Mount with anti-fade mounting medium

Cell Type-Specific Considerations:

• HepG2 cells show strong cytoplasmic LDHA staining

• HeLa and MCF7 cells display distinctive cytoplasmic patterns

• Adjust antibody concentration based on LDHA expression levels in different cell types

The search results indicate that LDHA antibodies detect the protein predominantly in the cytoplasm, consistent with its role in glycolysis .

What technical considerations are important when using LDHA antibodies for flow cytometry?

Flow cytometry with LDHA antibodies requires specialized techniques for intracellular targets:

Sample Preparation:

• For intracellular LDHA detection, proper fixation and permeabilization are critical

• Cell concentration recommendation: 10^6 cells in 100 μl suspension

Antibody Selection and Quantity:

• Use antibodies validated specifically for flow cytometry

• Recommended antibody amount: 0.25 μg per 10^6 cells in 100 μl suspension

• Consider using directly conjugated antibodies to eliminate secondary antibody steps

Protocol Optimization:

• Fixation Options:

  • Paraformaldehyde (1-4%) for structural preservation

  • Methanol/ethanol for enhanced permeabilization and epitope exposure

• Permeabilization Methods:

  • Saponin (0.1-0.5%): Reversible, gentle permeabilization

  • Triton X-100 (0.1-0.5%): More aggressive permeabilization

  • Commercial permeabilization buffers specifically designed for flow cytometry

• Blocking Step:

  • Use 5-10% serum or 1-3% BSA to reduce non-specific binding

  • Match serum species to secondary antibody host

• Controls Required:

  • Unstained cells

  • Isotype controls (matching the primary antibody isotype)

  • FMO (Fluorescence Minus One) controls

  • Positive controls (HepG2 cells are mentioned as positive for LDHA in flow cytometry)

• Compensation:

  • Particularly important for multicolor panels including LDHA

  • Use single-color controls for each fluorophore

Analysis Considerations:

• Gating strategies should account for expected cytoplasmic distribution

• Evaluate median fluorescence intensity rather than just percent positive

• Consider using stimulated vs. unstimulated samples to detect metabolic shifts affecting LDHA levels

Flow cytometry allows quantitative assessment of LDHA expression at the single-cell level, enabling correlation with other cellular parameters and heterogeneity analysis within populations.

How can LDHA antibodies be used to study protein-protein interactions and post-translational modifications?

LDHA antibodies can be powerful tools for investigating protein-protein interactions and post-translational modifications using several advanced techniques:

Co-Immunoprecipitation (Co-IP):

• Several LDHA antibodies in the search results are validated for immunoprecipitation (IP)

• Protocol outline:

  • Lyse cells in non-denaturing buffer

  • Pre-clear lysate with protein A/G beads

  • Incubate cleared lysate with LDHA antibody (typically 2-5 μg)

  • Add protein A/G beads to capture antibody-antigen complexes

  • Wash extensively to remove non-specific binding

  • Elute and analyze by Western blot for interacting partners

Proximity Ligation Assay (PLA):

• Enables in situ detection of protein-protein interactions

• Combine LDHA antibody with antibody against potential interacting partner

• Species of primary antibodies must be different

• Detection of close proximity (<40 nm) indicates interaction

Post-Translational Modification Analysis:

• Phosphorylation: The search results mention antibodies specific for phospho-Tyr10 on LDHA

• Workflow:

  • Immunoprecipitate LDHA using a total LDHA antibody

  • Probe with anti-phosphotyrosine antibody (1:2000 dilution mentioned)

  • Alternatively, use phospho-specific LDHA antibodies directly

Mass Spectrometry Approaches:

• Immunoprecipitate LDHA from cells/tissues

• Perform tryptic digestion

• Analyze peptides by LC-MS/MS to identify:

  • Interacting proteins

  • Post-translational modifications

  • Sequence variants

Chromatin Immunoprecipitation (ChIP):

• For investigating nuclear translocation and potential DNA interactions

• While LDHA is primarily cytoplasmic, reports of nuclear functions exist

• Combine with LDHA antibodies for novel insights

The search results indicate that LDHA can form tetramers and interact with other proteins, making these techniques valuable for understanding its functional complexes and regulation .

What are the methodological approaches for using LDHA antibodies in CRISPR/Cas9 gene-edited samples?

LDHA antibodies are essential tools for validating and characterizing CRISPR/Cas9 gene-edited samples:

Validation of LDHA Knockout Efficiency:

• Western blot analysis using validated LDHA antibodies

• The search results describe CRISPR/Cas9 targeting of LDHA with specific guide RNAs

• Target sequence example: 5′-TAGGAGGCTATGGACTTGCAGCA-3′

Knockout/Knockdown Validation Protocol:

• Design and synthesize sgRNA targeting LDHA exons

• Transfect/electroporate cells with Cas9 and sgRNA

• Isolate clonal populations

• Extract protein from wild-type and knockout cells

• Perform Western blot with LDHA antibody:

  • Load equal amounts of protein (30 μg recommended)

  • Include appropriate loading controls (β-actin commonly used)

  • Compare band intensity between wild-type and knockout samples

Phenotypic Characterization:

• Use LDHA antibodies to correlate knockout efficiency with phenotypic changes

• Applications for phenotypic analysis:

  • Immunofluorescence to assess metabolic changes in single cells

  • Flow cytometry for population analysis

  • Cell viability, colony formation, and apoptosis assays were used in the context of T-ALL research

Rescue Experiments:

• Reintroduce wild-type or mutant LDHA to knockout cells

• The search results describe a human LDHA transgene rescue approach

• Confirm expression using species-specific or epitope-tagged antibodies

• Example from research: Human LDHA antibody detection of rescue construct alongside endogenous mouse LDHA

Cross-Species Considerations:

• When using human LDHA to rescue mouse knockouts, species-specific antibodies are valuable

• The search results describe using anti-human LDHA antibody (1:2000 dilution) to detect the transgene product separately from mouse proteins

These approaches provide robust validation of CRISPR/Cas9 editing and enable detailed functional analysis of LDHA in various experimental systems.

How can researchers investigate LDHA's role in cellular metabolism using antibodies combined with metabolic assays?

Investigating LDHA's role in metabolism requires integrating antibody-based detection with functional metabolic assays:

Combined Immunoblotting and Metabolic Profiling:

• Western Blot Analysis:

  • Quantify LDHA expression levels using validated antibodies (1:500-1:3000 dilution)

  • Correlate with expression of related metabolic enzymes

  • Example protocol from search results:

    • 5-20% SDS-PAGE separation

    • Transfer to nitrocellulose membrane at 150 mA

    • Block with 5% non-fat milk/TBS

    • Incubate with anti-LDHA antibody overnight at 4°C

• Metabolic Inhibition Studies:

  • Treat cells with LDHA inhibitors (e.g., oxamate mentioned in search results)

  • Monitor effects on:

    • Cell viability (MTT assay)

    • Colony formation

    • Apoptosis

    • Cell cycle progression

• ROS Production Analysis:

  • The search results describe mitochondrial ROS assays in conjunction with LDHA inhibition

  • Correlate ROS levels with LDHA expression or activity

Immunofluorescence Combined with Metabolic Imaging:

• Co-localize LDHA with mitochondrial markers

• Use fluorescent metabolic probes alongside LDHA immunostaining

• Analyze subcellular distribution in response to metabolic challenges

Flow Cytometry with Metabolic Parameters:

• Simultaneously assess LDHA levels and metabolic indicators:

  • Mitochondrial membrane potential

  • Glucose uptake

  • ROS production

• Protocol considerations:

  • Use 0.25 μg antibody per 10^6 cells

  • Carefully coordinate fixation/permeabilization to preserve both LDHA epitopes and metabolic indicators

Tissue-Level Analysis:

• Immunohistochemistry to map LDHA expression in tissues with different metabolic profiles

• Serial section analysis to correlate LDHA with other metabolic markers

• The search results mention IHC protocols using 1:100-1:400 dilutions for LDHA antibodies

These integrated approaches enable researchers to establish functional relationships between LDHA expression/activity and metabolic phenotypes in various experimental systems.

What are the technical challenges and solutions when using LDHA antibodies for electron microscopy studies?

Electron microscopy (EM) studies with LDHA antibodies present unique technical challenges and require specialized approaches:

Challenges:

• Epitope Preservation: Standard EM fixation protocols (glutaraldehyde/osmium tetroxide) can mask or destroy LDHA epitopes

• Penetration Issues: Gold-conjugated antibodies must access intracellular targets

• Specificity Concerns: Background can be problematic in the high-contrast EM environment

• Quantification Difficulties: Relating gold particle density to protein abundance

Methodological Solutions:

• Optimal Fixation Protocol:

  • Mild fixation with low concentration paraformaldehyde (0.5-2%)

  • Avoid or minimize glutaraldehyde (0-0.1%)

  • Consider using specialized fixatives designed for immunogold EM

• Immunogold Labeling Approach:

  • Pre-embedding vs. post-embedding techniques

  • The search results describe an immunogold EM protocol:

    • Incubate sections on grids with 0.05 M glycine in PBS (5 min)

    • Block with 5% BSA in PBS (15 min)

    • Incubate with primary antibody (e.g., goat anti-LDHA diluted 1:50) for 1 hour

    • Wash with PBS (5× for 5 min each)

    • Label with gold-conjugated secondary antibody (10 nm gold particles)

    • Wash to remove unbound conjugates

• Controls for EM Immunogold:

  • Omit primary antibody (essential negative control)

  • Use tissue from LDHA knockout models

  • Include gradient of known LDHA expression samples

• Double-Labeling Strategies:

  • Combine LDHA detection with other metabolic enzymes

  • Use different sized gold particles (e.g., 5 nm vs. 10 nm)

  • Consider sequential labeling protocols

• Quantification Approaches:

  • Systematic random sampling

  • Stereological principles

  • Normalization to membrane length or organelle volume

The search results describe successful electron microscopy localization of LDHA using immunogold techniques, demonstrating the feasibility of this advanced application while highlighting the need for careful optimization .