GFOD2 Antibody

What is GFOD2 and why is it important for research?

GFOD2 (Glucose-Fructose Oxidoreductase Domain Containing 2) is a protein encoded by the GFOD2 gene (GeneID: 81577, HGNC: 28159) . It has several alternative names in the literature including FLJ39316, FLJ23802, MGC11335, and UNQ9430/PRO34691 . The protein contains oxidoreductase domains that suggest enzymatic activity related to glucose-fructose metabolism, making it potentially significant in metabolic pathways and lipid metabolism research . While the complete function of GFOD2 remains under investigation, its study may provide insights into metabolic disorders, cellular redox processes, and related pathologies. The observed molecular weight of human GFOD2 is approximately 42 kDa in western blot analyses .

What applications are commonly used with GFOD2 antibodies?

GFOD2 antibodies are validated for multiple experimental applications in molecular and cellular biology research. The most commonly validated applications include:

• Western Blot (WB): Used to detect GFOD2 protein in cell lysates, typically at dilutions of 1:500-1:2000

• Enzyme-Linked Immunosorbent Assay (ELISA): Utilized for quantitative detection, with recommended dilutions of 1:500-1:3000

• Immunohistochemistry (IHC): For tissue section analysis, with optimal dilutions ranging from 1:50-1:200

• Immunocytochemistry/Immunofluorescence (ICC/IF): For cellular localization studies, typically used at 1-4 μg/ml

Several antibodies show cross-validation across multiple applications, providing researchers flexibility in experimental design. For instance, some commercially available antibodies are validated for WB, ELISA, and IHC simultaneously, enabling complementary approaches to GFOD2 detection and characterization .

What species reactivity is available for GFOD2 antibodies?

GFOD2 antibodies demonstrate varying species reactivity profiles, which is an important consideration when designing experiments. Based on the available data:

Human-reactive GFOD2 antibodies are most prevalent and extensively validated across multiple applications. For cross-species studies, it's essential to select antibodies specifically validated for the target species. When conducting evolutionary or comparative studies, researchers should verify the sequence homology of the epitope region across species to ensure reliable detection and minimize false negatives due to species-specific variations in the GFOD2 protein sequence .

What are the optimal storage conditions for maintaining GFOD2 antibody activity?

Proper storage of GFOD2 antibodies is critical for maintaining their reactivity and specificity. For short-term storage (up to one month), antibodies should be kept at 4°C in their original buffer . For long-term storage, aliquoting and freezing at -20°C is recommended to avoid repeated freeze-thaw cycles that can degrade antibody performance .

The standard storage buffer for most commercial GFOD2 antibodies consists of PBS (pH 7.3) supplemented with 0.02% sodium azide and 40-50% glycerol . This formulation provides stability by preventing microbial growth and protecting protein structure during freeze-thaw processes. According to recommendations from multiple suppliers, GFOD2 antibodies typically maintain activity for approximately 12 months when stored properly .

Key storage recommendations:

• Create small working aliquots to minimize freeze-thaw cycles

• Maintain sterile conditions when handling antibodies

• Document date of first use and number of freeze-thaw cycles

• Avoid exposure to direct light, especially for fluorophore-conjugated antibodies

• Monitor stability through consistent positive controls in experiments

How should I validate the specificity of GFOD2 antibodies?

Validation of GFOD2 antibody specificity is crucial for experimental reliability. A comprehensive validation strategy should include multiple approaches:

• Western Blot Analysis: Verify detection of a single band at the expected molecular weight of approximately 42 kDa using human cell lines such as Jurkat cells . Multiple bands may indicate non-specific binding or detection of isoforms.

• Protein Array Testing: Select antibodies that have been validated against protein arrays containing the target protein along with hundreds of non-specific proteins to confirm selectivity .

• Epitope Analysis: Verify that the antibody recognizes a specific epitope sequence. For example, some GFOD2 antibodies are developed against the recombinant protein corresponding to the amino acid sequence: VGSAGRLVARGADLYGQKNSATQEELLLRDSLAVGAGLPEQGPQDVPLLYLKGMVYMVQALRQSFQ .

• Knockout/Knockdown Controls: When possible, validate using GFOD2 knockout or knockdown samples to confirm absence of signal.

• Peptide Competition Assay: Pre-incubate the antibody with increasing concentrations of the immunizing peptide to demonstrate signal reduction proportional to peptide concentration.

What are the recommended dilutions for GFOD2 antibodies across different applications?

Optimal dilutions for GFOD2 antibodies vary by application and specific antibody characteristics. Based on manufacturer recommendations and research protocols, the following dilution ranges should be considered:

When working with a new batch of antibody or a different experimental system, it is advisable to perform a dilution series to determine the optimal concentration that maximizes specific signal while minimizing background. Antibody concentration may need adjustment based on expression levels of GFOD2 in different tissues or cell types.

How do polyclonal and monoclonal GFOD2 antibodies differ in research applications?

Polyclonal and monoclonal GFOD2 antibodies offer distinct advantages and limitations that significantly impact experimental outcomes:

Polyclonal GFOD2 Antibodies:

• Most commercially available GFOD2 antibodies are polyclonal, primarily raised in rabbits

• Recognize multiple epitopes on the GFOD2 protein, potentially increasing detection sensitivity

• Show greater tolerance to minor protein denaturation or modification

• May exhibit batch-to-batch variation requiring additional validation

• Optimal for initial protein characterization and applications where high sensitivity is prioritized over absolute specificity

Monoclonal GFOD2 Antibodies:

• Recognize a single epitope, providing higher specificity but potentially lower sensitivity

• Offer greater consistency between batches, enhancing experimental reproducibility

• May be more vulnerable to epitope masking or modification

• Can be developed using recombinant methods as described in recent literature on antibody development

For critical quantitative analyses, researchers should consider using monoclonal antibodies or validate polyclonal antibodies across multiple lots. When detecting low abundance GFOD2 protein, polyclonal antibodies may offer advantages due to their typically higher avidity. The choice between polyclonal and monoclonal should be guided by the specific research question, required level of specificity, and need for reproducibility across long-term studies.

What factors affect GFOD2 antibody stability and how can they be mitigated?

GFOD2 antibody stability is influenced by multiple physical and chemical factors that can compromise experimental reproducibility. Understanding and addressing these factors is essential for robust research outcomes.

The stability of antibodies, including those targeting GFOD2, can be affected by:

• Temperature Fluctuations: Repeated freeze-thaw cycles can lead to antibody denaturation. This can be mitigated by storing antibodies in small working aliquots to minimize the number of freeze-thaw cycles .

• pH Changes: Extreme pH conditions can irreversibly alter antibody conformation. Most GFOD2 antibodies are formulated in PBS at pH 7.3, which provides optimal stability .

• Protein Aggregation: Antibody aggregation reduces effective concentration and increases non-specific binding. The addition of 40-50% glycerol in storage buffers helps prevent aggregation during freeze-thaw cycles .

• Oxidation: Oxidative damage can affect antibody binding sites. Minimizing exposure to strong oxidants and light can reduce this risk.

• Microbial Contamination: Preservatives like 0.02% sodium azide are included in commercial formulations to prevent microbial growth .

Recent research in antibody engineering has demonstrated that computational methods can be used to identify and modify regions prone to aggregation, potentially improving stability . These approaches analyze B-factors, conformational entropy, and molecular dynamics to predict destabilizing features in the antibody structure. Such engineering approaches could be applied to enhance the stability of GFOD2 antibodies for challenging experimental conditions.

How can I troubleshoot inconsistent results when using GFOD2 antibodies in Western blot?

Inconsistent results when using GFOD2 antibodies in Western blot analyses can stem from several sources. A structured troubleshooting approach helps identify and address these issues:

Problem: No signal or weak signal detection

• Potential causes: Insufficient protein loading, inefficient transfer, antibody degradation, low target expression

• Solutions:

  • Verify protein loading using total protein stains or housekeeping proteins

  • Optimize transfer conditions (time, voltage, buffer composition)

  • Use fresh antibody aliquots and validate activity with positive controls

  • Extend exposure time or use more sensitive detection systems

Problem: Multiple unexpected bands

• Potential causes: Non-specific binding, cross-reactivity, protein degradation, post-translational modifications

• Solutions:

  • Increase blocking time or agent concentration (5% BSA or milk)

  • Optimize antibody dilution (typically 1:500-1:2000 for GFOD2)

  • Include protease inhibitors during sample preparation

  • Verify with another GFOD2 antibody recognizing a different epitope

Problem: High background

• Potential causes: Insufficient blocking, excessive antibody concentration, extended exposure

• Solutions:

  • Increase blocking time or agent concentration

  • Dilute primary and secondary antibodies further

  • Include additional washing steps with higher Tween-20 concentration (0.1-0.3%)

Problem: Variable band intensity between experiments

• Potential causes: Inconsistent loading, transfer efficiency variation, antibody degradation

• Solutions:

  • Implement standardized loading controls

  • Use internal reference standards across blots

  • Consider using Jurkat cells as positive controls, which have been validated for GFOD2 detection

Documentation of all experimental parameters and systematic variation of individual factors can help identify the source of inconsistency.

How do I interpret unexpected molecular weights when detecting GFOD2?

When working with GFOD2 antibodies, researchers sometimes observe bands at molecular weights that differ from the expected 42 kDa . These variations require careful interpretation rather than immediate dismissal as non-specific binding:

Potential explanations for unexpected molecular weights:

• Post-translational modifications: Glycosylation, phosphorylation, or other modifications can significantly alter the apparent molecular weight of GFOD2. The protein name itself (Glucose-Fructose Oxidoreductase Domain Containing 2) suggests possible involvement in glycosylation processes.

• Alternative splicing: The GFOD2 gene may produce multiple protein isoforms through alternative splicing, resulting in proteins of different molecular weights. UniProt lists several secondary accession numbers (Q69YL9, Q6UXX6, Q7L648, Q8TE86, Q9BQ07, R4GNG5) that may represent different isoforms or database entries .

• Proteolytic processing: In vivo or during sample preparation, proteolytic cleavage can generate fragments of the full-length protein.

• Protein complexes: Incomplete denaturation may result in detection of GFOD2 in protein complexes, appearing at higher molecular weights.

To distinguish between these possibilities:

• Compare results across different cell types or tissues

• Use denaturing agents of varying strengths

• Employ multiple antibodies targeting different epitopes of GFOD2

• Consider additional protease inhibitors during sample preparation

• Perform peptide competition assays to verify specificity

The observed molecular weight should be documented and compared with literature reports to build a more complete understanding of GFOD2 biology.

What controls should be included when using GFOD2 antibodies in research?

Implementing appropriate controls is essential for generating reliable and interpretable data with GFOD2 antibodies. A comprehensive control strategy should include:

Positive Controls:

• Jurkat cell lysates have been specifically validated for GFOD2 detection and serve as excellent positive controls for Western blot applications

• Human tissue samples with known GFOD2 expression for IHC applications

• Recombinant GFOD2 protein at known concentrations for quantitative applications

Negative Controls:

• Secondary antibody only (omitting primary GFOD2 antibody) to assess non-specific binding of the detection system

• Isotype controls matched to the GFOD2 antibody's isotype (typically IgG)

• When available, GFOD2 knockout or knockdown samples

Specificity Controls:

• Peptide competition/blocking experiments using the immunizing peptide or recombinant GFOD2 protein

• Testing on samples from multiple species when using antibodies claimed to have cross-species reactivity

Procedural Controls:

• Loading controls for Western blot (housekeeping proteins or total protein stains)

• Standardized reference samples included across multiple experiments to normalize inter-experimental variation

• Dilution series to demonstrate signal proportionality to antigen concentration

Implementing these controls enables more confident data interpretation and facilitates troubleshooting when unexpected results occur. For publication-quality research, documentation of these controls is increasingly required by journals and reviewers.

How can new antibody engineering approaches be applied to improve GFOD2 antibody performance?

Recent advances in antibody engineering offer opportunities to enhance GFOD2 antibody performance for demanding research applications. These approaches can address limitations in current antibodies:

Computational stability engineering: Recent research demonstrates that computational methods can identify and modify regions prone to aggregation in antibodies . By analyzing B-factors, conformational entropy, and molecular dynamics, researchers can predict destabilizing features in antibody structure. For GFOD2 antibodies, this approach could yield variants with improved thermal stability and reduced aggregation propensity, particularly valuable for techniques requiring harsh conditions or long-term storage.

Golden Gate-based dual-expression systems: A novel method for rapid screening of recombinant monoclonal antibodies uses a Golden Gate-based dual-expression vector system allowing in-vivo expression of membrane-bound antibodies . This approach could accelerate the development of high-affinity monoclonal GFOD2 antibodies by enabling simultaneous expression of heavy and light chains from a single vector, streamlining the screening process.

Epitope optimization: Careful selection of immunogens based on protein structural analysis can target highly specific regions of GFOD2. Using recombinant proteins corresponding to specific amino acid sequences, as seen in some commercial GFOD2 antibodies, researchers can generate antibodies with higher specificity .

Format diversification: Beyond traditional antibody formats, engineering GFOD2-specific single-chain variable fragments (scFvs) or antigen-binding fragments (Fabs) could provide advantages for certain applications, such as improved tissue penetration for immunohistochemistry or reduced background in multiplex immunofluorescence.

These emerging approaches represent the frontier of antibody technology that could be applied to develop next-generation GFOD2 detection reagents with enhanced performance characteristics.

What are the future directions for GFOD2 antibody research?

As our understanding of GFOD2 biology continues to evolve, several promising research directions emerge for antibody development and application:

• Expanded application validation: While current GFOD2 antibodies are validated for standard techniques like WB, ELISA, and IHC, future work should focus on validating these antibodies for emerging technologies such as proximity ligation assays, mass cytometry, and super-resolution microscopy.

• Isoform-specific antibodies: Development of antibodies that can specifically distinguish between potential GFOD2 isoforms would enable more nuanced studies of isoform-specific functions and expression patterns.

• Species cross-reactivity expansion: Broader validation of cross-species reactivity would facilitate comparative studies across model organisms, enhancing translational research potential.

• Post-translational modification detection: Engineering antibodies specifically designed to detect phosphorylated, glycosylated, or otherwise modified forms of GFOD2 could provide insights into regulatory mechanisms.

• Integration with rapid antibody development pipelines: Utilizing emerging technologies like the Golden Gate-based dual-expression system could accelerate the development of highly specific monoclonal antibodies against GFOD2 .

• Stability enhancement through computational design: Application of computational stability engineering approaches could yield more robust GFOD2 antibodies suitable for challenging experimental conditions .