Gapdh1 Antibody, Biotin conjugated

What is GAPDH and why is it used as a research target?

GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) is a catalytic enzyme that plays a key role in glycolysis. It exists as a tetramer of identical 37-kDa subunits and catalyzes the reversible reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphophate in the presence of NADPH . GAPDH is one of the most commonly used loading control proteins for immunodetection applications like Western blot due to its ubiquitous expression in most tissues . This widespread expression makes it an ideal housekeeping protein for normalization in quantitative experiments.

Beyond its glycolytic function, GAPDH is involved in numerous cellular processes including DNA replication, DNA repair, nuclear RNA export, membrane fusion, and microtubule bundling . This multifunctionality has expanded GAPDH's research applications beyond simple loading controls to studies on apoptosis, neurodegeneration, and cancer biology.

What are the advantages of biotin-conjugated GAPDH antibodies?

Biotin-conjugated GAPDH antibodies offer several distinct advantages in research applications:

• Enhanced sensitivity: The biotin-avidin/streptavidin system provides signal amplification due to the multiple binding sites on avidin/streptavidin molecules.

• Versatility in detection methods: Biotin-conjugated antibodies can be detected using various streptavidin-conjugated reporter molecules (HRP, fluorophores, gold particles), allowing flexibility in experimental design .

• Compatibility with multiplexing: Biotin-conjugated antibodies can be combined with differently labeled primary antibodies for simultaneous detection of multiple proteins.

• Reduced background: The high affinity and specificity of the biotin-streptavidin interaction (Kd = 10^-15 M) minimizes non-specific binding.

• Stability: Biotin conjugation typically does not significantly affect antibody stability or specificity when properly performed .

What applications are biotin-conjugated GAPDH antibodies validated for?

Biotin-conjugated GAPDH antibodies have been validated for multiple research applications as indicated in the product specifications. According to the search results, these antibodies are validated for:

When selecting a biotin-conjugated GAPDH antibody, researchers should verify that the specific product has been validated for their application of interest .

What species reactivity can be expected with GAPDH antibodies?

GAPDH is highly conserved across species, but antibody reactivity can vary significantly based on the epitope targeted and antibody type. Based on the search results, the following species reactivities are documented:

• Goat polyclonal anti-GAPDH biotin antibody: Reacts with GAPDH from human, mouse, rat, goat, rabbit, chicken, hamster, bovine, and swine .

• Mouse monoclonal anti-GAPDH biotin antibody (1A10): Specifically detects human GAPDH but is negative for mouse and rat GAPDH .

• Rabbit recombinant monoclonal anti-GAPDH biotin antibody (RM114): Reacts with human, mouse, and rat GAPDH, with potential cross-reactivity to other species .

When planning experiments involving multiple species, researchers should carefully select an antibody with appropriate cross-reactivity or species-specific detection properties based on their experimental design requirements.

How can biotin-conjugated GAPDH antibodies be used to study non-glycolytic functions of GAPDH?

GAPDH has numerous non-glycolytic functions that can be investigated using biotin-conjugated antibodies through various advanced techniques:

• Nuclear Translocation Studies: GAPDH can translocate to the nucleus during apoptosis and other cellular processes. Immunofluorescence combined with subcellular fractionation can track GAPDH localization changes using biotin-conjugated antibodies and fluorescent streptavidin conjugates .

• Protein-Protein Interaction Analysis: Co-immunoprecipitation (Co-IP) using biotin-conjugated GAPDH antibodies can pull down GAPDH-associated proteins including the amyloid precursor protein, polyglutamine tracts of Huntingtin, actin, and tubulin . The biotin tag allows gentle elution conditions preserving interaction integrity.

• Chromatin Immunoprecipitation (ChIP): For studying GAPDH's role in gene regulation, biotin-conjugated antibodies can be used in ChIP assays to identify DNA regions bound by GAPDH .

• GAPDH in Neurodegeneration Models: Biotin-conjugated antibodies can detect changes in GAPDH expression, localization, and post-translational modifications in Alzheimer's and Huntington's disease models .

• Cell Death Pathway Investigation: Using flow cytometry with biotin-conjugated GAPDH antibodies can help quantify changes in GAPDH expression during apoptosis across cell populations .

These applications extend well beyond GAPDH's traditional role as a loading control and leverage the versatility of biotin conjugation for detection flexibility.

What considerations are important when using GAPDH as a loading control in disease models?

While GAPDH is widely used as a loading control, several important considerations must be addressed when working with disease models:

• Variable Expression in Disease States: GAPDH expression can be altered in certain pathological conditions. For example, hypoxia and diabetes can increase GAPDH expression in specific cell types . Researchers should validate GAPDH stability in their particular disease model.

• Tissue-Specific Expression Differences: Although expressed in most tissues, GAPDH levels can vary significantly between tissue types . When comparing different tissues, alternative loading controls or total protein normalization might be more appropriate.

• Altered Expression in Cancer: GAPDH is involved in cancer-related processes including neuronal apoptosis in cancer models . This makes it potentially unsuitable as a loading control in certain cancer studies.

• Neurodegenerative Disease Complications: GAPDH has direct interactions with proteins involved in Alzheimer's and Huntington's diseases, including amyloid precursor protein and Huntingtin . These interactions may alter its detection or expression in neurodegeneration models.

• Subcellular Redistribution: Under stress conditions, GAPDH can redistribute between cellular compartments, potentially affecting whole-cell lysate measurements even when total expression remains constant .

For critical quantitative studies, researchers should consider validating multiple housekeeping genes or using total protein normalization methods (like Ponceau S or Stain-Free technologies) as alternatives to GAPDH.

How can researchers optimize multiplexing protocols with biotin-conjugated GAPDH antibodies?

Multiplexing with biotin-conjugated GAPDH antibodies requires careful optimization to prevent cross-reactivity and signal interference:

• Sequential Detection Protocol:

  • Apply primary antibodies from different host species (e.g., rabbit anti-target protein and biotin-conjugated mouse anti-GAPDH)

  • Use species-specific secondary antibody for the first primary (e.g., anti-rabbit-Alexa 488)

  • Block any remaining binding sites with excess unconjugated secondary antibody

  • Apply fluorophore-conjugated streptavidin (e.g., streptavidin-Alexa 647) to detect biotin-GAPDH

  • This prevents cross-detection between detection systems

• Optimized Dilutions Table:

• Spectral Separation: When using fluorescent detection, ensure adequate spectral separation between fluorophores to minimize bleed-through. Biotin-conjugated GAPDH can be detected with far-red fluorophores (e.g., Alexa 647), while target proteins use green or red channels.

• Signal Balancing: GAPDH's abundant expression may require higher dilution of biotin-conjugated antibody compared to target proteins of interest to prevent signal saturation .

• Validation Controls: Include single-stained controls to verify specificity and absence of cross-reactivity between detection systems.

These optimization steps ensure reliable simultaneous detection of GAPDH and proteins of interest in multiplexed experimental setups.

What is the optimal Western blot protocol for biotin-conjugated GAPDH antibodies?

The following optimized Western blot protocol for biotin-conjugated GAPDH antibodies incorporates best practices for high sensitivity and specificity:

Sample Preparation and Gel Electrophoresis:

• Prepare whole cell lysates in RIPA buffer with protease inhibitors

• Quantify protein concentration using Bradford or BCA assay

• Load 10-20 μg of protein per lane (GAPDH is abundant at ~37 kDa)

• Separate proteins using 10-12% SDS-PAGE gel

Transfer and Blocking:

• Transfer proteins to PVDF or nitrocellulose membrane

• Block membrane in 5% non-fat dry milk or 3% BSA in TBST for 1 hour at room temperature

• For BSA blocking, use biotin-free BSA to prevent interference with detection system

Primary Antibody Incubation:

• Dilute biotin-conjugated GAPDH antibody to 1:1000-1:5000 in blocking buffer

• Incubate membrane overnight at 4°C with gentle rocking

• Wash 3-5 times with TBST, 5 minutes each

Detection:

• Incubate membrane with streptavidin-HRP (1:2000-1:5000) in blocking buffer for 1 hour at room temperature

• Wash 3-5 times with TBST, 5 minutes each

• Develop using ECL substrate and image using appropriate detection system

• Expected band size: 37 kDa (GAPDH monomer)

Optimization Tips:

• For weaker signals, extending streptavidin-HRP incubation to 90 minutes can improve sensitivity

• If background is high, increase washing steps or duration

• GAPDH typically gives strong signals; avoid overexposure by starting with higher antibody dilutions (1:5000)

• For simultaneous detection with other proteins, consider using fluorescently-labeled streptavidin instead of HRP for multiplex detection

How should researchers validate the specificity of biotin-conjugated GAPDH antibodies?

Validating antibody specificity is crucial for reliable research results. For biotin-conjugated GAPDH antibodies, consider these comprehensive validation methods:

• Positive and Negative Control Samples:

  • Positive controls: Cell lines known to express GAPDH (most mammalian cell lines)

  • Negative controls: Species-specific negative samples (if antibody is human-specific, use mouse samples as negative control)

  • Recombinant GAPDH protein as positive control for absolute specificity verification

• Molecular Weight Verification:

  • GAPDH should appear as a single band at approximately 37 kDa in Western blot

  • Multiple bands may indicate degradation, post-translational modifications, or non-specific binding

• Knockdown/Knockout Validation:

  • Compare GAPDH detection in wild-type cells versus GAPDH siRNA knockdown cells

  • Signal should decrease proportionally to knockdown efficiency

• Peptide Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide (if available)

  • Specific signals should be blocked by peptide competition

• Cross-Validation with Different Antibody Clones:

  • Compare results with a non-biotin conjugated GAPDH antibody from a different clone

  • Agreement between different antibodies increases confidence in specificity

• Immunoprecipitation-Mass Spectrometry:

  • Perform IP with biotin-conjugated GAPDH antibody

  • Analyze pulled-down proteins by mass spectrometry

  • GAPDH should be the predominant protein identified

• Blocking Endogenous Biotin:

  • Include avidin/streptavidin blocking step before applying biotin-conjugated antibody

  • This controls for potential endogenous biotin interference

Thorough validation ensures that experimental results reflect true GAPDH biology rather than artifacts from non-specific antibody binding.

What protocol modifications are needed for immunohistochemistry with biotin-conjugated GAPDH antibodies?

Immunohistochemistry (IHC) with biotin-conjugated GAPDH antibodies requires special considerations due to both endogenous GAPDH expression and endogenous biotin in tissues:

Optimized IHC Protocol for Biotin-Conjugated GAPDH Antibodies:

• Sample Preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Process and embed in paraffin

  • Section at 4-5 μm thickness

  • Mount on positively charged slides

• Deparaffinization and Antigen Retrieval:

  • Deparaffinize in xylene and rehydrate through graded alcohols

  • Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Maintain at 95-98°C for 20 minutes

  • Cool to room temperature for 20 minutes

• Critical Endogenous Biotin Blocking (Essential Step):

  • Block endogenous biotin using a commercial avidin/biotin blocking kit

  • Apply avidin solution for 15 minutes

  • Rinse with PBS

  • Apply biotin solution for 15 minutes

  • Rinse with PBS

• Peroxidase and Protein Blocking:

  • Block endogenous peroxidase with 3% hydrogen peroxide for 10 minutes

  • Block non-specific binding with 2-5% normal serum from the same species as the secondary antibody for 30 minutes

• Primary Antibody Incubation:

  • Dilute biotin-conjugated GAPDH antibody to 1:100-1:300 in antibody diluent

  • Incubate overnight at 4°C in a humidified chamber

• Detection:

  • Rinse slides with PBS (3 × 5 minutes)

  • Apply streptavidin-HRP (1:500) for 30 minutes at room temperature

  • Rinse with PBS (3 × 5 minutes)

  • Develop with DAB substrate for 3-10 minutes while monitoring microscopically

  • Counterstain with hematoxylin, dehydrate, and mount

• Controls and Validation:

  • Include tissue known to express GAPDH as positive control

  • Include a negative control by omitting primary antibody

  • Consider using a non-biotin detection system on a sequential section for comparison

This protocol addresses the critical challenge of endogenous biotin in tissues, which can lead to false positive results if not properly blocked .

How can researchers troubleshoot inconsistent results with biotin-conjugated GAPDH antibodies?

When encountering inconsistent results with biotin-conjugated GAPDH antibodies, systematically address these common issues:

No Signal or Weak Signal:

High Background or Non-specific Binding:

Unexpected Band Patterns/Localization:

Inconsistent Results Between Experiments:

Methodological Verification Steps:

• Run side-by-side comparisons with non-biotin GAPDH antibody

• Test on samples with known GAPDH expression levels

• Verify antibody functionality with dot blot before complex applications

• Consider alternative detection systems if biotin-based methods consistently fail

How do physiological and pathological conditions affect GAPDH expression and antibody detection?

GAPDH expression can be significantly altered under various physiological and pathological conditions, which directly impacts its utility as a housekeeping control and experimental target:

Physiological Conditions Affecting GAPDH Expression:

• Hypoxia: Oxygen deprivation upregulates GAPDH expression in many cell types as cells shift toward glycolytic metabolism. This can lead to overestimation of normalization when using GAPDH as a loading control in hypoxia studies .

• Cell Proliferation Status: Rapidly proliferating cells typically show higher GAPDH expression compared to quiescent cells, reflecting increased metabolic demands.

• Tissue-Specific Variation: GAPDH expression levels vary naturally between different tissues. Brain, liver, and muscle tissues tend to express higher levels than connective tissues .

• Developmental Stages: GAPDH expression can change during development, with embryonic tissues often showing different expression patterns compared to adult tissues.

Pathological Conditions Affecting GAPDH Detection:

• Neurodegenerative Diseases: In Alzheimer's and Huntington's diseases, GAPDH interacts with disease-specific proteins (amyloid precursor protein and Huntingtin, respectively), potentially affecting its detection or cellular distribution .

• Diabetes: Diabetic conditions can increase GAPDH expression in certain cell types, making it potentially unsuitable as a reference gene in diabetes research .

• Cancer: GAPDH is involved in cancer-related processes including apoptosis regulation. Its expression is often elevated in many cancer types, creating normalization challenges in cancer research .

• Inflammation: Inflammatory conditions can alter GAPDH expression levels, again potentially confounding its use as a housekeeping gene in inflammation studies.

• Oxidative Stress: GAPDH is sensitive to oxidative modifications, which can affect both its function and antibody epitope recognition.

These variations highlight the importance of validating GAPDH stability in each specific experimental context before using it as a reference gene or analytical target .

What are the considerations for using biotin-conjugated GAPDH antibodies in flow cytometry?

Flow cytometry with biotin-conjugated GAPDH antibodies presents unique challenges and optimization opportunities:

Protocol Optimization for Flow Cytometry:

• Cell Preparation and Fixation:

  • Thoroughly fix and permeabilize cells as GAPDH is predominantly intracellular

  • Recommended fixation: 4% paraformaldehyde for 15 minutes at room temperature

  • Recommended permeabilization: 0.1% Triton X-100 or saponin-based buffers for 10 minutes

• Endogenous Biotin Blocking:

  • Include avidin/biotin blocking step before antibody application

  • Treat cells with unconjugated avidin (10-15 minutes) followed by excess biotin (10-15 minutes)

  • This prevents false positive signal from endogenous biotin in cells

• Antibody Concentration Optimization:

  • Titrate biotin-conjugated GAPDH antibody (typically start at 1:100-1:300)

  • Determine optimal concentration by signal-to-noise ratio assessment

  • For multicolor panels, higher dilutions may be needed due to GAPDH abundance

• Streptavidin-Fluorophore Selection:

  • Choose fluorophore based on cytometer configuration and panel design

  • For multicolor panels, consider bright fluorophores like PE, APC or BV421

  • Titrate streptavidin-fluorophore conjugate to minimize background

• Gating Strategy Considerations:

  • Include FMO (Fluorescence Minus One) controls to set proper gates

  • Expect relatively tight GAPDH distribution in homogeneous populations

  • Cell cycle phases may show different GAPDH expression levels

Potential Pitfalls and Solutions:

Advanced Applications:

• GAPDH can serve as a cell viability marker in certain contexts

• Changes in GAPDH levels can be monitored during apoptosis or cell stress

• Cell sorting based on GAPDH expression can isolate populations with different metabolic states

These considerations ensure reliable detection of GAPDH by flow cytometry while minimizing technical artifacts.

How can researchers study GAPDH's role in neurodegenerative diseases using biotin-conjugated antibodies?

GAPDH's involvement in neurodegenerative diseases can be investigated through several specialized approaches using biotin-conjugated antibodies:

Co-localization Studies in Disease Models:

• Use biotin-conjugated GAPDH antibodies with streptavidin-fluorophores alongside antibodies against disease-specific proteins (amyloid-β, tau, α-synuclein, huntingtin)

• Analyze co-localization in:

  • Patient-derived tissue samples

  • Animal models of neurodegeneration

  • iPSC-derived neuronal cultures

• Quantify co-localization coefficients using confocal microscopy

Protein-Protein Interaction Analysis:

• Utilize biotin-conjugated GAPDH antibodies for co-immunoprecipitation of disease-relevant protein complexes

• Protocol modifications for neurodegenerative research:

  • Use gentle lysis buffers to preserve weak interactions

  • Consider crosslinking before lysis to capture transient interactions

  • Elute with biotin for native complex isolation

• Analyze precipitated complexes by Western blot or mass spectrometry

Subcellular Fractionation Analysis:

• GAPDH accumulates in the nucleus during neuronal apoptosis in neurodegenerative conditions

• Fractionate neuronal samples into cytoplasmic, nuclear, and membrane fractions

• Use biotin-conjugated GAPDH antibodies to track redistribution between compartments

• Compare fractionation patterns between healthy and diseased samples

Post-translational Modification Studies:

• GAPDH undergoes S-nitrosylation, oxidation, and other modifications in neurodegeneration

• Combine biotin-switch technique for detecting S-nitrosylated GAPDH with biotin-conjugated GAPDH antibodies

• Compare modification patterns in control vs. disease models

• Correlate modifications with localization changes and protein interactions

Therapeutic Target Validation:

• Use biotin-conjugated GAPDH antibodies to assess effects of potential neuroprotective compounds on:

  • GAPDH nuclear translocation

  • GAPDH-mediated apoptosis

  • Interaction with disease-specific proteins

• Quantify changes in GAPDH localization or interactions following treatment

Experimental Design Considerations:

• Include age-matched controls for all neurodegenerative disease samples

• Consider brain region-specific variations in GAPDH expression and function

• Account for potential interference from disease-related protein aggregates

• Validate findings across multiple disease models and patient samples

These approaches leverage the versatility of biotin-conjugated GAPDH antibodies to investigate its non-glycolytic roles in neurodegeneration, potentially revealing novel therapeutic targets.

What are the optimal storage conditions for biotin-conjugated GAPDH antibodies?

Proper storage and handling are essential for maintaining the activity and specificity of biotin-conjugated GAPDH antibodies. Based on the search results, the following storage recommendations should be followed:

• Temperature Requirements:

  • Most biotin-conjugated GAPDH antibodies should be stored at -20°C for long-term storage

  • Some formulations may be stored at 4°C in the dark, as specified for certain products

  • Avoid repeated freeze-thaw cycles which can degrade antibody activity and biotin conjugation

• Buffer Conditions:

  • Typical storage buffers include:

    • PBS with 0.05% sodium azide

    • PBS with 1% BSA, 50% glycerol and 0.09% sodium azide

  • The presence of protein stabilizers (BSA) and cryoprotectants (glycerol) helps maintain antibody integrity

  • Sodium azide serves as a preservative but is incompatible with HRP-based detection systems

• Aliquoting Recommendations:

  • Upon receipt, divide antibody into small single-use aliquots

  • Use sterile microcentrifuge tubes for aliquoting

  • Minimize air exposure during aliquoting to prevent oxidation of biotin

• Stability Considerations:

  • Typical shelf life ranges from 12-24 months when properly stored

  • Working dilutions should be prepared fresh and used within 24 hours

  • Avoid exposure to strong light which may damage both the antibody and biotin conjugate

• Shipping and Temporary Storage:

  • If received on ice, transfer immediately to recommended storage conditions

  • Brief storage at 4°C (less than 1 week) is generally acceptable

  • Monitor for signs of degradation after any deviation from recommended storage

• Documentation Practices:

  • Record receipt date, lot number, and expiration date

  • Document freeze-thaw cycles

  • Consider including performance validation after extended storage

Following these storage guidelines will help maintain optimal antibody performance throughout the product's intended shelf life .