GAPDH Recombinant Monoclonal Antibody

What is GAPDH and why is it a significant target for antibody development?

GAPDH is a multifunctional enzyme that primarily catalyzes the conversion of D-glyceraldehyde 3-phosphate (G3P) into 3-phospho-D-glyceroyl phosphate during glycolysis. It exists as a tetramer of identical 37-kDa subunits and is ubiquitously expressed in most tissues . Beyond its metabolic role, GAPDH participates in numerous cellular processes including DNA replication, DNA repair, nuclear RNA export, membrane fusion, and microtubule bundling . Its consistent expression pattern across most cell types makes it an ideal loading control for Western blot and other protein quantification techniques . Additionally, GAPDH functions as a component of the GAIT (gamma interferon-activated inhibitor of translation) complex, which mediates interferon-gamma-induced transcript-selective translation inhibition during inflammation processes .

What advantages do recombinant monoclonal antibodies offer over conventional antibodies for GAPDH detection?

Recombinant monoclonal antibodies provide several significant advantages when studying GAPDH:

• Enhanced specificity and sensitivity for target detection

• Consistent performance with minimal lot-to-lot variation

• Animal origin-free formulations in many cases

• Broader immunoreactivity due to the larger rabbit immune repertoire

These antibodies are produced using in vitro expression systems by cloning specific antibody DNA sequences from immunoreactive rabbits, followed by screening individual clones to select optimal candidates . For GAPDH specifically, the high conservation of this protein across species makes antibody consistency particularly valuable for comparative studies and reproducible quantification.

What species reactivity can researchers expect from GAPDH recombinant monoclonal antibodies?

GAPDH recombinant monoclonal antibodies demonstrate broad cross-reactivity across multiple species due to the high evolutionary conservation of GAPDH. Validated reactivity typically includes:

• Human (including cell lines like HeLa, CACO-2, CCRF-CEM, A549)

• Mouse (brain and liver tissues)

• Rat (brain and liver tissues)

• Chicken

• Monkey

• Zebrafish

What are the validated applications for GAPDH recombinant monoclonal antibodies?

GAPDH recombinant monoclonal antibodies have been validated for multiple applications in molecular and cellular biology research:

These applications have been verified across multiple cell lines and tissue types, including human cancer tissues such as laryngeal squamous cell carcinoma, renal clear cell carcinoma, and ovarian serous cancer .

How should researchers design Western blot experiments using GAPDH as a loading control?

For optimal Western blot results using GAPDH as a loading control, follow these methodological guidelines:

• Sample preparation:

  • Use 30 μg of total protein per lane for consistent detection

  • Ensure complete lysis using appropriate detergents

  • Include protease inhibitors to prevent degradation

• Gel electrophoresis:

  • Use 5-20% SDS-PAGE gels for optimal separation

  • Run at 70V for stacking gel and 90V for resolving gel (2-3 hours)

• Transfer and blocking:

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

  • Block with 5% non-fat milk/TBS for 1.5 hours at room temperature

• Antibody incubation:

  • Incubate with anti-GAPDH antibody at 0.5 μg/mL overnight at 4°C

  • Wash thoroughly with TBS-0.1% Tween (3 times, 5 minutes each)

  • Incubate with HRP-conjugated secondary antibody at 1:5000 dilution

• Detection:

  • Develop using enhanced chemiluminescence (ECL)

  • Expect a specific band at approximately 36 kDa

It's important to note that GAPDH expression can vary under certain experimental conditions such as hypoxia, cancer progression, or apoptosis, so validation of consistent expression is necessary when studying these processes.

What protocol should be followed for immunofluorescence detection of GAPDH?

For optimal immunofluorescence detection of GAPDH, implement this methodological protocol:

• Cell preparation:

  • Culture cells on appropriate coverslips

  • Fix with 4% paraformaldehyde

  • Permeabilize with appropriate buffer

• Antigen retrieval (if needed):

  • Use enzyme antigen retrieval reagents (e.g., for 15 minutes)

• Staining procedure:

  • Block with 10% goat serum

  • Incubate with GAPDH antibody at 5 μg/mL overnight at 4°C

  • Wash thoroughly

  • Incubate with fluorophore-conjugated secondary antibody (e.g., DyLight®488) at 1:500 dilution

  • Counterstain nuclei with DAPI

• Controls:

  • Include secondary antibody-only controls

  • Consider GAPDH knockdown controls for specificity validation

This protocol has been validated in various cell lines including A549 cells, demonstrating specific cytoplasmic detection of GAPDH with occasional nuclear localization depending on cellular conditions .

How does GAPDH conservation impact antibody specificity across evolutionary distant species?

GAPDH is one of the most evolutionarily conserved proteins, which significantly impacts antibody cross-reactivity patterns:

These patterns indicate that while GAPDH is broadly conserved, specific epitopes may vary. This property can be leveraged to develop antibodies that selectively recognize GAPDH from particular phylogenetic groups, making them valuable tools for studying evolutionary relationships or for discriminating between host and pathogen GAPDH in infection models .

How can researchers leverage GAPDH antibody cross-reactivity for comparative studies?

The broad cross-reactivity of GAPDH antibodies enables several sophisticated comparative research approaches:

• Evolutionary studies:

  • Compare GAPDH epitope conservation across species

  • Analyze structural differences in GAPDH between phylogenetic groups

  • Study functional domain conservation through differential antibody binding

• Host-pathogen interaction studies:

  • Use differential reactivity profiles to distinguish host from pathogen GAPDH

  • Investigate GAPDH as a virulence factor in bacterial pathogens

  • Study GAPDH surface expression in bacterial species

• Multi-species experimental systems:

  • Apply the same antibody across different model organisms for consistent detection

  • Create standardized loading controls for cross-species protein expression studies

  • Develop quantitative comparisons of GAPDH expression levels between species

These approaches utilize the combination of conservation and epitope variation to gain insights into both evolutionary relationships and functional divergence of GAPDH across taxa .

What common issues occur when using GAPDH as a loading control, and how can they be addressed?

Despite its popularity as a loading control, several challenges can arise when using GAPDH in quantitative experiments:

• Variable expression under experimental conditions:

  • Problem: GAPDH expression can change during hypoxia, cancer progression, and apoptosis

  • Solution: Validate GAPDH stability under your specific experimental conditions; consider multiple loading controls in parallel

• Saturation and non-linear response:

  • Problem: GAPDH's high abundance can lead to signal saturation

  • Solution: Perform titration experiments; use short exposure times; consider loading less protein

• Molecular weight overlap:

  • Problem: GAPDH (36-37 kDa) may overlap with similarly sized target proteins

  • Solution: Use pre-stained markers; strip and reprobe membranes sequentially

• Non-specific bands:

  • Problem: Additional bands may appear besides the expected GAPDH band

  • Solution: Optimize antibody dilution; increase washing stringency; use monoclonal antibodies for higher specificity

Addressing these issues methodically ensures more reliable quantification when using GAPDH as a reference protein.

How should researchers validate a new lot of GAPDH recombinant monoclonal antibody?

Proper validation of new antibody lots is crucial for experimental reproducibility. Follow this comprehensive validation protocol:

• Western blot validation:

  • Run positive control lysates (e.g., HeLa cells)

  • Test multiple dilutions (1:500, 1:1000, 1:5000)

  • Verify single band detection at expected molecular weight (36-37 kDa)

  • Compare signal intensity and background with previous lot

• Immunocytochemistry validation:

  • Perform staining on well-characterized cell lines

  • Verify expected subcellular localization pattern

  • Check for background and non-specific staining

• Quantitative assessment:

  • Perform side-by-side comparison with previous lot

  • Prepare standard curves using serial dilutions

  • Calculate detection sensitivity and linear range

• Documentation:

  • Record lot number, validation date, and experimental conditions

  • Archive validation results for future reference

If significant differences are observed between lots, contact the manufacturer for technical support and consider protocol adjustments.

Beyond loading controls, how can GAPDH recombinant monoclonal antibodies investigate non-glycolytic functions?

GAPDH has numerous non-glycolytic functions that can be studied using specialized experimental approaches:

• Nuclear translocation studies:

  • Use immunofluorescence with subcellular fractionation to track GAPDH movement between cytoplasm and nucleus

  • Apply co-immunoprecipitation to identify nuclear binding partners

  • Employ chromatin immunoprecipitation to investigate DNA interactions

• Protein-protein interaction analysis:

  • Perform co-immunoprecipitation using GAPDH antibodies

  • Use proximity ligation assays to visualize interactions between GAPDH and targets like amyloid precursor protein

  • Apply FRET approaches to study dynamic interactions

• Cytoskeletal interaction studies:

  • Examine GAPDH co-localization with microtubules and actin

  • Study GAPDH's role in modulating CHP1-dependent microtubule associations

  • Investigate cytoskeletal organization during cellular stress

These advanced applications leverage the specificity of recombinant monoclonal antibodies to dissect GAPDH's diverse non-glycolytic functions in cellular processes and disease mechanisms.

How can researchers use GAPDH antibodies to study its role in neurodegeneration?

GAPDH has been implicated in several neurodegenerative disorders through interactions with disease-associated proteins. When investigating these connections, researchers should consider these methodological approaches:

• Protein aggregation studies:

  • Use sequential protein extraction to isolate soluble vs. aggregated GAPDH

  • Apply Western blot analysis on different fractions

  • Optimize sample preparation to preserve protein-protein interactions

• Co-localization analysis:

  • Perform double immunofluorescence for GAPDH and disease proteins (e.g., amyloid-β, Huntingtin)

  • Use super-resolution microscopy for detailed analysis

  • Apply proximity ligation assays to confirm direct interactions

• Functional assays in disease models:

  • Assess nuclear translocation of GAPDH during cellular stress

  • Evaluate GAPDH-mediated apoptotic signaling

  • Test compounds that disrupt pathological GAPDH interactions

GAPDH is reported to bind to proteins implicated in neurodegenerative diseases, including amyloid precursor protein (Alzheimer's disease) and the polyglutamine tracts of Huntingtin (Huntington's disease) . These methodological approaches enable investigation of GAPDH's complex roles in neurodegeneration mechanisms.

What methodological approaches can investigate GAPDH in the GAIT complex during inflammation?

GAPDH is a component of the GAIT complex, which mediates interferon-gamma-induced transcript-selective translation inhibition. To study this specialized function, researchers can employ these methodological approaches:

• Complex composition analysis:

  • Immunoprecipitate GAPDH under interferon-gamma treatment

  • Perform mass spectrometry on co-precipitated proteins

  • Validate interactions with known components through Western blot

• RNA-protein interaction studies:

  • Conduct RNA immunoprecipitation using GAPDH antibodies

  • Couple with RT-qPCR to identify target transcripts containing GAIT elements

  • Use crosslinking approaches for mapping interaction sites

• Functional studies:

  • Use siRNA knockdown of GAPDH followed by rescue with mutant versions

  • Assess impact on GAIT complex formation

  • Measure translation efficiency of GAIT element-containing reporters

• Localization analysis:

  • Track GAPDH redistribution during interferon-gamma response

  • Co-stain for other GAIT complex components

These approaches allow researchers to dissect GAPDH's specific contribution to translational regulation during inflammation, separating this function from its glycolytic role .