OGFOD2 Antibody

What is OGFOD2 and why is it relevant to researchers?

OGFOD2 is a 2-oxoglutarate and iron-dependent oxygenase domain-containing protein that functions as an oxidoreductase. This enzyme plays important roles in cellular metabolism and has been implicated in several biological processes. OGFOD2 belongs to the Gfo/Idh/MocA protein family and promotes matrix assembly . Research interest in OGFOD2 has increased due to its potential involvement in metabolic disorders such as diabetes and obesity, as dysregulation of this protein affects cellular energy balance and function . Additionally, genetic studies have associated OGFOD2 with schizophrenia, as noted in research by the Schizophrenia Working Group of the Psychiatric Genomics Consortium .

To maintain antibody integrity and reactivity, proper storage is essential:

• Store antibodies at -20°C for long-term storage .

• For short-term use, some antibodies can be kept at 4°C but follow manufacturer recommendations .

• Aliquot antibodies upon receipt to avoid repeated freeze/thaw cycles that can damage antibody structure and function .

• For conjugated antibodies (e.g., FITC-labeled), protect from light exposure to prevent photobleaching .

• Most OGFOD2 antibodies are supplied in buffer containing 40-50% glycerol as a cryoprotectant, often with 0.02-0.03% sodium azide as a preservative .

The validity period for properly stored antibodies is typically 12 months from the date of receipt .

Which species reactivity do commercial OGFOD2 antibodies exhibit?

Species reactivity varies between different OGFOD2 antibody products:

• Human-specific antibodies are available from some suppliers .

• Broader reactivity antibodies that recognize human, mouse, and rat OGFOD2 are more common .

• Some products indicate predicted reactivity with additional species based on sequence homology, though these require validation .

When selecting an antibody, confirm the species reactivity matches your experimental model and consider validation data provided by the manufacturer for your species of interest.

What controls are essential when designing experiments with OGFOD2 antibodies?

Proper controls are critical for accurate interpretation of OGFOD2 antibody experiments:

• Unstained cells/tissue: Include to assess autofluorescence that may increase the population of false-positive cells .

• Negative controls: Utilize cell populations not expressing OGFOD2 to confirm target specificity of the primary antibody .

• Isotype controls: Include an antibody of the same class as the primary antibody but with no known specificity (e.g., non-specific control IgG) to assess undesirable background staining due to Fc receptor binding .

• Secondary antibody controls: For indirect staining methods, include samples treated with only labeled secondary antibody to detect non-specific binding .

• Blocking peptide controls: Several manufacturers offer recombinant OGFOD2 protein antigens that can be used in competitive binding assays to confirm antibody specificity . For example, Novus Biologicals offers a recombinant protein antigen (NBP1-82254PEP) specifically designed as a blocking peptide for their OGFOD2 antibody .

• Positive controls: Consider using recombinant OGFOD2 protein in Western blot applications as a positive control .

How can researchers optimize Western blot protocols for OGFOD2 detection?

For optimal OGFOD2 detection in Western blot experiments:

• Sample preparation: OGFOD2 has an observed molecular weight of approximately 32-39 kDa . Use appropriate lysis buffers to ensure complete protein extraction.

• Gel selection: 10-12% polyacrylamide gels are suitable for resolving proteins in this molecular weight range.

• Protein loading: Begin with 20-30 μg of total protein per lane, adjusting based on expression levels in your sample.

• Transfer conditions: Standard transfer protocols for proteins of this size are appropriate (e.g., 100V for 1-2 hours or 30V overnight).

• Blocking: Use 5% non-fat milk or BSA in TBST to reduce background.

• Antibody dilution: Follow manufacturer recommendations, typically in the range of 1:500-1:2000 . For Novus Biologicals antibody, 0.4 μg/ml is recommended .

• Incubation time: Typically overnight at 4°C for primary antibody and 1-2 hours at room temperature for secondary antibody.

• Washing: Perform thorough washing (3-5 times for 5-10 minutes) with TBST between antibody incubations.

• Detection method: Both ECL-based chemiluminescence and fluorescence-based methods are suitable.

What strategies can improve specificity when using OGFOD2 antibodies in immunohistochemistry?

To enhance specificity in immunohistochemistry applications:

• Antigen retrieval optimization: Test both heat-induced (citrate buffer, pH 6.0 or EDTA buffer, pH 9.0) and enzymatic methods to determine optimal epitope exposure.

• Blocking optimization: Use 10% normal serum from the same host species as the secondary antibody to reduce background. Importantly, ensure the normal serum is NOT from the same host species as the primary antibody to avoid non-specific signals .

• Antibody titration: Test a range of dilutions around the manufacturer's recommendation (1:20-1:500) to determine the optimal signal-to-noise ratio for your tissue type.

• Incubation conditions: Compare room temperature versus 4°C incubation, and various incubation times (1 hour to overnight).

• Secondary antibody selection: Use highly cross-adsorbed secondary antibodies to minimize cross-reactivity.

• Chromogen/fluorophore selection: Consider tissue autofluorescence when selecting fluorophores; DAB may be preferable for tissues with high autofluorescence.

• Multi-step detection systems: For low-expression targets, consider using amplification systems such as tyramide signal amplification.

How do polyclonal and monoclonal OGFOD2 antibodies compare in research applications?

Most commercially available OGFOD2 antibodies are polyclonal , which affects their application characteristics:

Polyclonal OGFOD2 antibodies:

• Recognize multiple epitopes on the target protein, potentially providing stronger signal

• May offer higher sensitivity for detection of low-abundance targets

• Show greater tolerance to minor changes in the antigen (denaturation, fixation, etc.)

• Batch-to-batch variation may require revalidation

• Typically derived from rabbit hosts for OGFOD2

Theoretical advantages of monoclonal antibodies (though less common for OGFOD2):

• Recognition of a single epitope for higher specificity

• More consistent results across experiments and batches

• Reduced background from non-specific binding

• Better suited for quantitative applications

• May have lower sensitivity than polyclonals unless using detection amplification

When selecting between antibody types, consider your experimental requirements for specificity versus sensitivity, and the importance of reproducibility for your research application.

How can researchers validate OGFOD2 antibodies for novel applications beyond manufacturer recommendations?

For extending OGFOD2 antibody applications beyond manufacturer-validated protocols:

• Knockout/knockdown validation: Generate OGFOD2-knockout or knockdown models to serve as negative controls, confirming signal specificity .

• Overexpression systems: Create positive controls by overexpressing tagged OGFOD2 in relevant cell lines to verify antibody detection capabilities.

• Epitope mapping: Determine which region of OGFOD2 the antibody recognizes to predict potential cross-reactivity and effects of fixation or denaturation.

• Cross-application validation: If an antibody works well in Western blot, systematically test and optimize it for immunoprecipitation, ChIP, or other applications.

• Biophysics-informed modeling: As described in recent research, combining "biophysics-informed modeling and extensive selection experiments holds broad applicability beyond antibodies, offering a powerful toolset for designing proteins with desired physical properties" .

• Independent antibody comparison: Compare results using different antibodies targeting distinct OGFOD2 epitopes. Multiple manufacturers offer OGFOD2 antibodies raised against different immunogens:

  • Recombinant human 2-oxoglutarate and iron-dependent oxygenase domain-containing protein 2 (1-186AA)

  • ARPEVYDSLQDAALAPEFLAVTEYSVSPDADLKGLLQRLETVSEEKRIYRVPVFTAPFCQALLEELEHFEQSDMPKGRPNTMNNYG

  • Recombinant fragment corresponding to 5-173aa of human OGFOD2

What analytical techniques can complement OGFOD2 antibody-based detection?

To strengthen research findings beyond antibody-dependent methods:

• Mass spectrometry: For unbiased protein identification and quantification of OGFOD2 and its interaction partners.

• RNA-seq or qPCR: To correlate protein expression with transcript levels.

• Recombinant expression systems: Using tagged versions of OGFOD2 for detection via the tag rather than antibody-based detection of the native protein.

• CRISPR-Cas9 genome editing: For visualization of endogenous OGFOD2 by knock-in of fluorescent proteins.

• Protein interaction assays: Such as proximity ligation assay (PLA) or FRET to study OGFOD2's functional interactions.

• Enzymatic activity assays: To directly measure OGFOD2's 2-oxoglutarate and iron-dependent oxygenase activity rather than just protein presence.

• Computational modeling: Predicting structure-function relationships based on sequence and available structural data.

How can researchers address common issues when working with OGFOD2 antibodies?

Problem: High background in immunostaining

• Solution: Optimize blocking (use 10% normal serum) , increase washing steps, and titrate antibody concentration. Consider using highly cross-adsorbed secondary antibodies to reduce non-specific binding.

Problem: Multiple bands in Western blot

• Solution: OGFOD2 has two isoforms produced by alternative splicing , which may appear as multiple bands. Verify molecular weight (expected ~32-39kDa) and use recombinant OGFOD2 protein as a positive control .

Problem: Weak or no signal

• Solution: Check protein expression levels in your sample, optimize antigen retrieval for fixed samples, increase antibody concentration or incubation time, and ensure proper sample preparation to preserve the epitope.

Problem: Inconsistent results across experiments

• Solution: Standardize protocols, use the same antibody lot when possible, include consistent positive and negative controls, and maintain detailed records of experimental conditions.

What considerations are important when using OGFOD2 antibodies across different sample types?

Different sample preparations require specific optimization:

Cell lines:

• Consider fixation method (4% paraformaldehyde vs. methanol) based on epitope location

• Optimize permeabilization to access intracellular epitopes

• Evaluate endogenous expression levels before experiments

Tissue sections:

• Paraffin-embedded tissues require appropriate antigen retrieval methods

• Fresh frozen tissues may preserve epitopes better but with reduced morphology

• Consider tissue-specific autofluorescence when choosing detection methods

Protein lysates:

• Select lysis buffers that effectively solubilize OGFOD2 while preserving the epitope

• Include protease inhibitors to prevent degradation

• Consider native vs. denaturing conditions based on the epitope recognized

How are OGFOD2 antibodies being used in metabolic disease research?

OGFOD2's involvement in glucose metabolism makes it relevant for metabolic disease research :

• Diabetes investigations: OGFOD2 antibodies can be used to study protein expression changes in models of insulin resistance and diabetes.

• Obesity research: Examining OGFOD2 expression and localization in adipose tissue and liver samples from obese vs. lean subjects.

• Metabolic pathway analysis: Studying OGFOD2's interaction with other metabolic enzymes and regulatory proteins.

• Drug discovery: Screening compounds that may modulate OGFOD2 function as potential therapeutic targets for metabolic disorders.

What role do OGFOD2 antibodies play in neuroscience research?

Given the association between OGFOD2 and schizophrenia , these antibodies have emerging applications in neuroscience:

• Expression profiling: Mapping OGFOD2 distribution in different brain regions and neural cell types.

• Genetic association studies: Correlating OGFOD2 variants with protein expression levels in patient-derived samples.

• Neurodevelopmental research: Examining OGFOD2's role in brain development and neural function.

• Pathology investigations: Comparing OGFOD2 expression and localization in neuropsychiatric disorder models.