OXNAD1 Antibody

What is OXNAD1 and what is its primary function in cellular processes?

OXNAD1 (Oxidoreductase NAD-binding domain-containing protein 1) is a protein that plays a key role in cellular defense against oxidative stress. It is involved in the response and regulation of oxidative stress processes, which contribute to various diseases including cancer, neurodegenerative disorders, and cardiovascular diseases . As an oxidoreductase with NAD-binding capabilities, OXNAD1 participates in redox reactions that are critical for maintaining cellular homeostasis under conditions of oxidative stress.

The protein's function makes it a significant target for research investigating cellular defense mechanisms and disease pathways. By studying OXNAD1, researchers can gain insights into how cells respond to oxidative damage and potentially develop therapeutic strategies targeting these pathways.

What are the typical molecular characteristics of OXNAD1 protein?

OXNAD1 has an observed molecular weight of approximately 34 kDa . The canonical human OXNAD1 protein spans amino acids 1-312, with the functional domain located between amino acids 18-312 . Some commercial antibodies are developed against specific fragments, such as amino acids 7-264 of human OXNAD1 .

Understanding these molecular characteristics is essential when selecting appropriate antibodies and designing experiments to detect and study OXNAD1. Researchers should be aware of the full-length protein size when analyzing Western blot results to confirm specific binding and avoid misinterpreting non-specific bands.

Which applications are most reliable for OXNAD1 detection using antibodies?

Based on commercial antibody specifications, OXNAD1 antibodies have been validated for several applications with varying recommended dilutions:

• Western Blot (WB): Commonly tested with dilutions ranging from 1:500-1:5000

• Enzyme-Linked Immunosorbent Assay (ELISA): Effective at dilutions of 1:2000-1:80000

• Immunohistochemistry (IHC): Validated at dilutions of 1:50-1:200

• Immunofluorescence (IF): Some antibodies are validated for this application

How can researchers validate the specificity of OXNAD1 antibodies?

Validating antibody specificity is critical for obtaining reliable research results. The most robust approach involves using knockout (KO) cell lines alongside isogenic parental controls . This method clearly demonstrates whether an antibody is specific to its target.

Methodology for OXNAD1 antibody validation:

• Select cell lines with sufficient expression of OXNAD1 (>2.5 log2(TPM+1) as RNA-level threshold)

• Generate OXNAD1 knockout cell lines using CRISPR-Cas9 or similar technologies

• Perform Western blot, immunoprecipitation, or immunofluorescence using both KO and parental cell lines

• A specific antibody will show signal in parental cells but no signal in KO cells

• Include positive controls (cell lines known to express OXNAD1) such as K562, U251, or A549 cell lines

According to studies evaluating commercial antibodies, approximately half of all antibodies do not meet manufacturer recommendations, highlighting the importance of validation .

What factors influence the variability in OXNAD1 antibody performance?

Several factors can affect OXNAD1 antibody performance:

• Antibody production method: Polyclonal antibodies (like most OXNAD1 antibodies) show batch-to-batch variability

• Epitope location: Antibodies targeting different regions of OXNAD1 may have varying accessibility depending on protein conformation

• Sample preparation: Fixation methods, buffer composition, and protein denaturation can affect epitope recognition

• Cross-reactivity: Some antibodies may cross-react with similar proteins, particularly between species

• Application-specific factors: An antibody that works well for Western blot may not perform equivalently in immunohistochemistry

Researchers should carefully evaluate antibody performance for their specific application and experimental conditions. The correlation between antibody performance in different applications (e.g., WB vs. IF) is not always strong, suggesting application-specific validation is necessary .

How does post-translational modification of OXNAD1 affect antibody detection?

While specific information about OXNAD1 post-translational modifications is limited in the provided search results, this is an important consideration for any protein detection. Post-translational modifications can potentially:

• Mask antibody epitopes, reducing detection efficiency

• Create new conformational states that alter antibody binding

• Change the apparent molecular weight of the protein in Western blots

• Affect subcellular localization, impacting immunofluorescence or IHC results

For OXNAD1 research, it's advisable to use antibodies raised against different regions of the protein and compare results. This approach helps identify whether post-translational modifications might be affecting detection in specific experimental conditions or disease states.

What are the optimal Western blot protocols for OXNAD1 detection?

Based on the commercial antibody specifications, the following Western blot protocol parameters are recommended for optimal OXNAD1 detection:

Sample preparation:

• Use cell lysates from tissues with known OXNAD1 expression (e.g., mouse large intestine, mouse lung, or human cell lines like K562, U251, or A549)

• Prepare whole cell lysates using standard lysis buffers containing protease inhibitors

Antibody concentrations and dilutions:

• Primary antibody:

  • PACO38730: 1:1000-1:5000 dilution

  • Abbexa antibody: 1:500-1:2000 dilution

  • Typically used at 4μg/ml for most commercial antibodies

• Secondary antibody: Anti-rabbit IgG at 1:10000 dilution

Detection system:

• Use standard chemiluminescence or fluorescence-based detection systems

• Expected band size: approximately 34 kDa

Controls:

• Positive control: Lysates from cells known to express OXNAD1

• Negative control: Ideally, OXNAD1 knockout cell lysates

This protocol should be optimized based on specific laboratory conditions and equipment.

How should researchers troubleshoot non-specific binding with OXNAD1 antibodies?

When encountering non-specific binding with OXNAD1 antibodies, researchers should implement the following troubleshooting strategies:

• Optimize antibody concentration: Test a dilution series to find the optimal concentration that maximizes specific binding while minimizing background

• Improve blocking conditions:

  • Increase blocking time or concentration

  • Try different blocking agents (BSA, milk, commercial blockers)

  • Add 0.1-0.3% Tween-20 to washing buffers

• Modify sample preparation:

  • Ensure complete protein denaturation for Western blot

  • Try different lysis buffers to improve protein extraction

  • Consider phosphatase or protease inhibitors if post-translational modifications are suspected

• Validate with knockout controls: The most definitive approach is comparing results between wild-type and OXNAD1 knockout samples

• Cross-validation with multiple antibodies: Use different OXNAD1 antibodies targeting distinct epitopes to confirm specificity

How should researchers interpret discrepant results between different OXNAD1 antibodies?

When facing discrepancies between different OXNAD1 antibodies, researchers should:

• Evaluate antibody validation status: Prioritize results from antibodies validated with knockout controls

• Consider epitope differences: Different antibodies may target distinct regions of OXNAD1, which could be differentially accessible depending on:

  • Protein conformation

  • Protein-protein interactions

  • Post-translational modifications

  • Splice variants

• Cross-reference with orthogonal methods:

  • Complement antibody-based detection with mRNA expression data

  • Use mass spectrometry for protein identification

  • Employ tagged OXNAD1 constructs as additional controls

• Statistical analysis: Apply appropriate statistical methods to quantify differences and determine if they are significant

• Report all findings transparently: Document all antibodies used, their catalog numbers, and any discrepancies observed to improve reproducibility in the field

Based on systematic antibody performance studies, approximately half of commercial antibodies may not meet manufacturer recommendations, explaining some discrepancies .

What statistical approaches are appropriate for analyzing OXNAD1 expression data?

When analyzing OXNAD1 expression data from antibody-based experiments, researchers should employ these statistical approaches:

• For Western blot densitometry:

  • Normalize OXNAD1 signal to appropriate loading controls

  • Use Student's t-test for two-group comparisons or ANOVA for multiple groups

  • Apply non-parametric tests (Mann-Whitney, Kruskal-Wallis) if data is not normally distributed

• For immunohistochemistry quantification:

  • Use standardized scoring systems for staining intensity

  • Apply chi-square tests for categorical data

  • Consider automated image analysis software for objective quantification

• For correlation analyses:

  • Use Pearson's correlation for normally distributed data

  • Apply Spearman's rank correlation for non-parametric data

  • Use McNemar test to evaluate concordance between different applications

• General considerations:

  • Account for technical replicates appropriately

  • Ensure biological replicates are sufficient for statistical power

  • Apply multiple testing correction (e.g., Bonferroni, FDR) when appropriate

  • Report exact p-values and effect sizes

Proper statistical analysis ensures reliable interpretation of OXNAD1 expression data across experimental conditions.

How is OXNAD1 being studied in cancer research?

OXNAD1 is emerging as a relevant protein in cancer research due to its role in oxidative stress regulation, which is frequently dysregulated in cancer cells. Researchers investigating OXNAD1 in cancer should consider:

• Expression analysis in cancer types:

  • Compare OXNAD1 expression between tumor and adjacent normal tissues

  • Analyze expression across cancer subtypes and stages

  • Correlate expression with patient outcomes

• Functional studies:

  • Knockout or knockdown OXNAD1 in cancer cell lines to assess effects on:

    • Proliferation and cell cycle

    • Apoptosis resistance

    • Migration and invasion

    • Resistance to oxidative stress-inducing therapies

• Recommended methodologies:

  • Western blot with validated antibodies (e.g., PACO38730 at 1:1000-1:5000 dilution)

  • IHC of tumor microarrays using antibodies validated for paraffin sections (1:50-1:200 dilution)

  • Cell line studies using lines with confirmed OXNAD1 expression (K562, U251, A549)

• Mechanistic investigations:

  • Study OXNAD1 interactome using co-immunoprecipitation

  • Analyze its role in ROS management pathways

  • Investigate connections to known cancer signaling pathways

OXNAD1 antibodies are valuable tools for advancing understanding of how oxidative stress response proteins contribute to cancer biology and potentially identifying new therapeutic targets .

What is the role of OXNAD1 in neurodegenerative and cardiovascular research?

OXNAD1 is relevant to both neurodegenerative and cardiovascular research due to its involvement in oxidative stress regulation, a critical factor in both disease categories :

Neurodegenerative Disease Research:

• Mechanistic relevance:

  • Oxidative stress is a hallmark of many neurodegenerative diseases including Alzheimer's, Parkinson's, and ALS

  • OXNAD1's role in oxidative stress defense makes it a potential factor in neuronal survival

• Research approaches:

  • Immunohistochemistry of brain tissues using validated antibodies (1:50-1:200)

  • Analysis of OXNAD1 expression in disease models versus controls

  • Correlation with markers of neurodegeneration and oxidative damage

Cardiovascular Disease Research:

• Pathophysiological context:

  • Oxidative stress contributes to atherosclerosis, hypertension, and heart failure

  • OXNAD1 may influence vascular endothelial cell response to oxidative insults

• Experimental strategies:

  • Study OXNAD1 expression in cardiac tissues under stress conditions

  • Assess impact of OXNAD1 modulation on cardiomyocyte survival

  • Investigate potential protective mechanisms against ischemia-reperfusion injury

• Technical considerations:

  • Use tissue-specific positive controls (e.g., mouse lung tissue)

  • Optimize fixation protocols for cardiac tissue immunohistochemistry

  • Consider species differences when using antibodies across model organisms

By studying OXNAD1 in these disease contexts, researchers may uncover new insights into the role of oxidative stress response systems and potentially identify novel therapeutic approaches targeting these pathways .