DHNAT2 Antibody

What is Dihydrofolate Reductase (DHFR) and why are antibodies against it important in research?

Dihydrofolate Reductase (DHFR) is a critical enzyme that catalyzes the conversion of dihydrofolate to tetrahydrofolate, an essential cofactor in nucleotide synthesis. DHFR antibodies are valuable research tools that enable detection, quantification, and localization of this enzyme in various experimental systems . These antibodies are particularly important for studies involving cell proliferation mechanisms, cancer research, and antimicrobial drug development, as DHFR is a key target for antifolate drugs.

What species cross-reactivity can be expected with commercially available DHFR antibodies?

Current DHFR antibodies often demonstrate robust cross-reactivity across human, mouse, and rat samples, making them versatile tools for comparative studies across these mammalian models . Western blot analyses have confirmed specific detection of DHFR at approximately 21 kDa in cell lines from multiple species, including human embryonic kidney (293T), human Burkitt's lymphoma (Raji), mouse myoblast (C2C12), and rat embryonic fibroblast (Rat-2) cell lines . When planning cross-species experiments, researchers should validate antibody performance in their specific experimental system as minor variations in epitope recognition may occur.

What are the recommended applications for DHFR antibodies in research?

DHFR antibodies have been validated for multiple applications including:

• Western blotting (under reducing conditions)

• Immunocytochemistry/Immunofluorescence

• Immunohistochemistry (on fixed tissues)

For Western blotting applications, optimal results are typically achieved using approximately 1 μg/mL of antibody followed by appropriate HRP-conjugated secondary antibodies . For immunocytochemistry applications, DHFR antibodies have successfully detected the target in fixed cell lines such as MCF-7 human breast cancer cells . Researchers should determine optimal dilutions for each application and cell/tissue type through titration experiments.

How can I optimize Western blot protocols when working with DHFR antibody to minimize background and maximize specific signal?

When optimizing Western blot protocols for DHFR antibody:

• Sample preparation: Use Immunoblot Buffer Group 1 for optimal results with DHFR antibody detection . Ensure complete cell lysis and protein denaturation.

• Reducing conditions: DHFR antibody performs optimally under reducing conditions that maintain the integrity of the epitope recognition site .

• Blocking optimization: Employ 3-5% BSA or milk protein in TBS-T for blocking, with initial testing to determine which provides lower background with your specific samples.

• Antibody concentration: Begin with 1 μg/mL and adjust based on signal-to-noise ratio in your experimental system .

• Detection system: HRP-conjugated secondary antibodies (such as Anti-Mouse IgG) provide reliable detection when paired with appropriate chemiluminescent substrates .

• Stripping and reprobing: If membrane stripping is necessary, use mild stripping buffers to preserve epitope integrity for subsequent detection.

These optimizations are particularly important when detecting DHFR in samples with lower expression levels or when quantitative comparisons are needed.

What criteria should be considered when selecting between monoclonal and polyclonal DHFR antibodies for specific research applications?

The selection between monoclonal and polyclonal DHFR antibodies should be based on:

For quantitative applications and long-term studies, monoclonal antibodies like those described in the literature (e.g., Clone #872442) offer advantages in consistency and reproducibility .

How can I validate the specificity of DHFR antibodies in my experimental system?

Thorough validation of DHFR antibodies should include:

• Positive controls: Use cell lines with confirmed DHFR expression, such as 293T or MCF-7 human cell lines .

• Knockout/knockdown controls: Compare antibody reactivity in wild-type versus DHFR-depleted samples (using CRISPR-Cas9 knockout or siRNA knockdown approaches).

• Molecular weight verification: Confirm detection at the expected molecular weight (~21 kDa for human DHFR) .

• Peptide competition assays: Pre-incubate antibody with purified DHFR or immunogen peptide to demonstrate signal specificity.

• Multiple antibody comparison: Use antibodies targeting different epitopes of DHFR to confirm concordant detection patterns.

• Orthogonal techniques: Correlate antibody-based detection with mRNA expression or enzymatic activity assays.

This comprehensive validation approach ensures reliable interpretation of experimental results and minimizes the risk of reporting artifacts.

What are the key considerations when designing immunofluorescence experiments with DHFR antibodies?

When designing immunofluorescence experiments with DHFR antibodies, researchers should consider:

• Fixation method: DHFR detection has been successful using immersion fixation methods that preserve epitope accessibility . Compare paraformaldehyde (4%) versus methanol fixation to determine optimal signal preservation.

• Permeabilization protocol: Titrate detergent concentration (e.g., 0.1-0.3% Triton X-100) to balance cell membrane permeabilization with antibody accessibility while preserving subcellular structures.

• Blocking parameters: Use 5-10% normal serum from the same species as the secondary antibody to minimize non-specific binding.

• Antibody concentration: Begin with manufacturer recommendations (typically 1-10 μg/mL) and optimize through serial dilutions.

• Counterstaining: Include nuclear counterstains (DAPI/Hoechst) and potentially cytoskeletal markers to provide context for DHFR localization.

• Controls: Include secondary-only controls and, ideally, DHFR-knockout/knockdown samples to validate specificity.

• Image acquisition parameters: Standardize exposure times, gain settings, and post-processing to allow for quantitative comparisons between experimental conditions.

For quantitative immunofluorescence, establish validation metrics including signal-to-noise ratio thresholds and coefficient of variation limits for technical replicates.

What approaches can be used to multiplex DHFR antibody detection with other antibodies in co-localization studies?

For successful multiplex staining with DHFR antibodies:

• Antibody species selection: Choose primary antibodies raised in different host species (e.g., mouse anti-DHFR with rabbit anti-target B) to enable simultaneous detection without cross-reactivity .

• Sequential staining protocol: If antibodies are from the same species, employ sequential staining with complete blocking steps between antibody pairs.

• Fluorophore selection: Choose fluorophores with minimal spectral overlap to reduce bleed-through (e.g., pair Alexa 488 with Alexa 647 rather than Alexa 555).

• Antibody concentration balancing: Adjust concentrations of each antibody independently to achieve comparable signal intensities for accurate co-localization analysis.

• Controls for each channel: Include single-stained controls for each antibody to confirm specificity and establish bleed-through compensation parameters.

• Quantitative co-localization metrics: Apply appropriate co-localization algorithms (Pearson's, Manders' coefficients) with statistical validation rather than relying solely on visual assessment.

Validation of multiplexed staining should include comparison to single-antibody staining patterns to confirm that antibody binding is not altered by the presence of additional antibodies.

How should researchers approach quantification of DHFR expression levels in Western blot and immunofluorescence data?

For rigorous quantification of DHFR expression:

Western Blot Quantification:

• Use loading controls (GAPDH, β-actin) appropriate for your experimental conditions

• Operate within the linear dynamic range of detection

• Normalize DHFR band intensity to loading control

• Include calibration standards when absolute quantification is needed

• Perform multiple technical and biological replicates (minimum n=3)

• Apply appropriate statistical tests based on data distribution

Immunofluorescence Quantification:

• Establish consistent exposure parameters across all experimental conditions

• Define objective criteria for positive cells versus background

• Quantify signal intensity using integrated density measurements

• Analyze sufficient fields of view (minimum 5-10 per condition)

• Use automated analysis workflows to reduce observer bias

• Account for cell-to-cell variability through appropriate sampling strategies

When comparing DHFR expression across different experimental conditions, researchers should report both the magnitude of change and the statistical significance of observed differences.

What approaches can help resolve discrepancies between antibody-based detection methods and other techniques for measuring DHFR levels?

When facing discrepancies between antibody-based and other detection methods:

• Technique-specific limitations assessment: Evaluate whether discrepancies may stem from post-translational modifications detected by antibodies but not by transcript-based methods.

• Epitope accessibility analysis: Consider whether protein conformation, complex formation, or localization might affect epitope recognition in different assays.

• Complementary methodology approach: Validate findings using orthogonal techniques such as:

  • Activity-based assays that measure DHFR enzymatic function

  • Mass spectrometry for absolute protein quantification

  • Proximity ligation assays for in situ protein interaction verification

• Time-course experiments: Investigate whether discrepancies reflect differences in mRNA versus protein half-life or translational regulation.

• Isoform-specific analysis: Determine whether antibodies detect specific DHFR isoforms that may be differentially expressed at the mRNA level.

How can DHFR antibodies be adapted for specialized applications such as ChIP-seq or proximity labeling techniques?

DHFR antibodies can be adapted for advanced applications through:

For ChIP-seq applications:

• Rigorous antibody validation for specificity using IP-Western and peptide competition assays

• Optimization of chromatin fragmentation conditions specific to the genomic regions of interest

• Development of strict quality control metrics for ChIP enrichment (signal-to-input ratios)

• Implementation of spike-in normalization for quantitative comparisons across samples

For proximity labeling techniques (BioID, APEX):

• Creation of DHFR fusion constructs with biotin ligases while preserving epitope recognition

• Validation that antibody recognition is not impaired by fusion protein expression

• Optimization of labeling conditions to capture transient interactions

• Development of appropriate controls to distinguish specific from non-specific interactions

For super-resolution microscopy:

• Selection of appropriate fluorophores with photostability suited to super-resolution techniques

• Determination of optimal antibody concentration to achieve sparse labeling for STORM/PALM

• Testing of both direct and indirect immunolabeling approaches to identify optimal resolution

These specialized applications require rigorous validation using positive and negative controls specific to each technique.

What are the latest developments in using antibody engineering to improve DHFR antibody performance in challenging research applications?

Recent innovations in antibody engineering that can enhance DHFR antibody performance include:

• Nanobody development: Single-domain antibody fragments derived from camelid heavy-chain antibodies offer advantages of smaller size (~15 kDa) and enhanced tissue penetration . This technology, similar to that used successfully for HIV targeting, could provide improved access to conformational epitopes of DHFR that might be inaccessible to conventional antibodies.

• Bispecific antibody formats: Engineering antibodies that simultaneously recognize DHFR and another target protein enables enhanced specificity for particular complexes or cellular compartments. This approach, inspired by techniques used in other fields, could significantly reduce background in co-localization studies.

• Tandem antibody configurations: Engineering antibodies in triple tandem formats has shown remarkable effectiveness in other systems, such as HIV neutralization, where this approach increased neutralization capability to 96% of diverse strains . Similar approaches could enhance DHFR detection sensitivity.

• Recombinant antibody production: E. coli-derived recombinant antibody fragments offer consistent lot-to-lot reproducibility and the ability to introduce site-specific modifications for conjugation or detection .

These technological advances offer promising avenues for researchers studying DHFR in contexts where conventional antibodies have limitations.

What are the most common sources of variability in DHFR antibody performance and how can they be addressed?

Common sources of variability in DHFR antibody performance include:

Implementing a quality control program with well-characterized positive control samples (e.g., 293T or MCF-7 cell lysates for DHFR) enables tracking of antibody performance over time and early detection of performance issues .

How can researchers distinguish between true negative results and technical failures when using DHFR antibodies in new experimental systems?

To distinguish true negatives from technical failures:

• Positive control implementation: Always include samples with known DHFR expression (e.g., 293T, Raji, C2C12, or Rat-2 cell lines) alongside experimental samples .

• Internal control verification: Confirm successful detection of housekeeping proteins in the same samples to verify successful protein extraction, transfer, and detection steps.

• Antibody functionality testing: Periodically validate antibody activity using established positive controls, especially before crucial experiments.

• Sensitivity determination: Establish the lower limit of detection through serial dilutions of positive control samples.

• Alternative detection methods: Confirm negative results using orthogonal approaches such as RT-qPCR for DHFR transcripts or enzymatic activity assays.

• Optimization for new systems: When working with new cell types or tissues, systematically optimize key parameters:

  • Sample preparation (lysis buffers, detergent concentration)

  • Antibody concentration (titration series)

  • Incubation conditions (time, temperature)

  • Detection systems (enhanced chemiluminescence reagents, exposure times)

This systematic approach helps distinguish biological absence of the target from technical limitations in detection.

How might DHFR antibodies be integrated into emerging single-cell analysis technologies?

Integration of DHFR antibodies into single-cell technologies can be approached through:

• Single-cell Western blotting: Adapting DHFR antibodies to microfluidic platforms that enable protein detection at single-cell resolution, providing insights into cell-to-cell variability in DHFR expression within heterogeneous populations.

• Mass cytometry (CyTOF): Conjugating DHFR antibodies with rare earth metals for simultaneous detection alongside dozens of other proteins in single cells, enabling comprehensive phenotyping that correlates DHFR expression with cell state.

• Spatial transcriptomics integration: Combining DHFR antibody detection with in situ transcriptomics to correlate protein expression with transcriptional profiles at single-cell resolution within tissue contexts.

• Microfluidic droplet encapsulation: Incorporating DHFR antibodies into droplet-based single-cell protein detection systems that can be paired with single-cell RNA-seq for multi-omic analysis.

• Live-cell antibody delivery: Developing cell-permeable DHFR antibody fragments for real-time tracking of DHFR dynamics in living cells, potentially using techniques similar to those employed in nanobody research .

These emerging applications require careful validation to ensure that sensitivity and specificity are maintained at the single-cell level, where target abundance may be significantly lower than in bulk samples.

What potential exists for using DHFR antibodies in therapeutic research contexts beyond their traditional role as research reagents?

DHFR antibodies have potential therapeutic research applications including:

• Targeted drug delivery systems: Conjugating DHFR antibodies with therapeutic payloads could enable specific targeting of cells with upregulated DHFR expression, such as rapidly proliferating cancer cells.

• Intrabody development: Engineering DHFR antibodies that can function within the cellular environment (intrabodies) could enable selective modulation of DHFR activity in specific cellular compartments or contexts.

• Proteolysis targeting chimeras (PROTACs): DHFR antibody-derived recognition domains could be incorporated into bifunctional molecules that recruit E3 ubiquitin ligases to DHFR, enabling selective degradation in specific contexts.

• Nanobody-based therapeutics: Drawing from advances in HIV research using nanobodies, similar approaches could be developed for DHFR targeting in specific disease states where DHFR activity contributes to pathology .

• Diagnostic imaging: Radiolabeled DHFR antibodies or fragments could potentially serve as imaging agents for visualizing tissues with high DHFR expression, similar to how other enzyme-targeted antibodies have been used in molecular imaging.

These therapeutic applications represent areas of active research where fundamental studies using DHFR antibodies as research tools can inform translational approaches.

How are advances in antibody technology likely to impact future DHFR research?

Emerging antibody technologies will transform DHFR research through:

• Synthetic antibody libraries: Phage-display and yeast-display technologies will enable rapid generation of DHFR antibodies with customized properties, potentially overcoming limitations of traditional immunization approaches. This parallels successes seen in other research areas where custom antibody arrays have enhanced diagnostic capabilities .

• Intracellular antibodies: Development of antibodies or antibody fragments that can function within living cells will enable real-time visualization and manipulation of DHFR in its native context.

• Allosteric modulators: Engineering antibodies that bind to allosteric sites on DHFR could provide new tools for modulating enzyme activity in research and potentially therapeutic contexts.

• Multiparametric detection systems: Integration of DHFR antibodies into multiplex detection platforms will enable simultaneous analysis of DHFR alongside other components of folate metabolism pathways, providing more comprehensive insights into metabolic regulation.

• AI-guided epitope selection: Computational approaches will enhance antibody design by predicting optimal epitopes for DHFR recognition with improved specificity and sensitivity.

These advances will expand the toolkit available for DHFR research, enabling more sophisticated investigations into its roles in normal physiology and disease states.

What novel applications of DHFR antibodies are emerging at the intersection of different research disciplines?

Cross-disciplinary applications of DHFR antibodies include:

• Systems biology approaches: Integration of DHFR antibody-based proteomics with metabolomics and transcriptomics to construct comprehensive models of folate metabolism regulation in different physiological and pathological states.

• Synthetic biology tools: Utilizing DHFR antibodies or derived binding domains as components in engineered cellular circuits, potentially as sensors or modulators of synthetic pathway activity.

• Nanomedicine applications: Incorporation of DHFR antibodies into nanoparticle-based delivery systems for targeted therapeutic delivery, building on advances in nanobody technology demonstrated in other fields .

• Environmental monitoring: Adaptation of DHFR antibodies for detecting antifolate compounds in environmental samples, with potential applications in ecological research and environmental health monitoring.

• Evolutionary biology studies: Using cross-species reactive DHFR antibodies to track evolutionary conservation and divergence of folate metabolism across different organisms .