DHFR Antibody

What is the optimal working concentration for DHFR antibodies across different applications?

When using DHFR antibodies, optimal concentrations vary by application and specific antibody clone. For immunohistochemistry on formalin-fixed paraffin-embedded tissues, antibodies like ab124814 have been validated at 1/1000 dilution (0.20 μg/mL) with heat-mediated antigen retrieval using Tris-EDTA buffer (pH 9.0) . For Western blotting, typical working concentrations range from 0.5-2 μg/mL, with successful detection demonstrated using 1 μg/mL of anti-DHFR monoclonal antibody on PVDF membranes . For immunocytochemistry, higher concentrations are often required, with 10 μg/mL showing good results in MCF-7 cells with a 3-hour room temperature incubation .

Always perform an antibody titration experiment (using serial dilutions from 0.1-20 μg/mL) against positive control samples to determine the optimal concentration that maximizes signal-to-noise ratio for your specific experimental system.

How can species cross-reactivity be verified when using DHFR antibodies?

DHFR antibodies vary in cross-reactivity across species. Several commercial antibodies detect human, mouse, and rat DHFR due to high sequence conservation. The MAB7934 antibody has validated reactivity with human, mouse, and rat DHFR in Western blot applications , while sc-377091 antibody is recommended for detection of DHFR from mouse, rat, and human origin .

To verify cross-reactivity:

• Perform Western blot analysis using positive control lysates from each species of interest

• Compare band sizes to the predicted molecular weight of DHFR (21 kDa)

• Include negative controls (DHFR-knockdown samples if available)

• Validate findings across multiple applications as cross-reactivity may differ between techniques

• Use recombinant DHFR proteins from different species as standards

Cross-reactivity should be experimentally confirmed for each new application, as detection sensitivity may vary between species despite sequence homology.

What controls are essential when using DHFR antibodies in experimental protocols?

Robust controls are critical for reliable DHFR antibody experiments:

Positive controls:

• Cell lines with known DHFR expression (293T, Raji, C2C12, and Rat-2 have been validated)

• Recombinant DHFR protein standards

• Tissues with established DHFR expression patterns

Negative controls:

• Primary antibody omission control

• Isotype-matched non-specific antibody control

• DHFR-knockdown or knockout samples when available

• Peptide competition assays to confirm specificity

Technical controls:

• Loading controls for Western blots (α-tubulin, GAPDH)

• Serial dilution of samples to confirm linear range of detection

• For immunohistochemistry, include normal tissues with varying DHFR expression levels

• For immunofluorescence, include autofluorescence controls

Implementing these controls allows proper interpretation of results and troubleshooting of unexpected findings across experimental systems.

What is the expected subcellular localization pattern when using DHFR antibodies?

DHFR exhibits dual localization in both the cytoplasm and mitochondria, with distribution patterns that may vary by cell type and physiological state . Immunofluorescence studies in MCF-7 human breast cancer cell lines show specific subcellular staining patterns that can be visualized using fluorescent-conjugated secondary antibodies and counterstaining with DAPI .

For accurate subcellular localization studies:

• Use confocal microscopy for better resolution of compartments

• Co-stain with mitochondrial markers (e.g., MitoTracker) and nuclear counterstains (DAPI)

• Compare patterns across multiple cell types and tissue samples

• Consider subcellular fractionation followed by Western blotting as a complementary approach

• Use multiple DHFR antibody clones to confirm localization patterns

The subcellular distribution of DHFR may shift under certain conditions, including cell cycle progression, metabolic stress, or drug treatments targeting folate metabolism pathways.

How can DHFR antibodies be optimized for immunoprecipitation experiments?

Successful DHFR immunoprecipitation requires careful optimization:

Recommended lysis buffer composition:

• 150 mM NaCl

• 5 mM EDTA

• 50 mM HEPES, pH 7.5

• 1.0% Triton X-100

• Protease inhibitor cocktail

Optimized procedure:

• Prepare cell lysates on ice to preserve protein integrity

• Pre-clear lysates with protein G-Sepharose to reduce non-specific binding

• Incubate with anti-DHFR antibody (3-5 μg per mg total protein) at 4°C overnight

• Add protein G-Sepharose 4 Fast Flow beads and incubate for 2-4 hours at 4°C

• Wash beads 3-5 times with lysis buffer

• Elute with SDS-PAGE sample buffer for analysis

Critical parameters:

• Maintain samples at 4°C throughout to prevent protein degradation

• Include negative controls (non-specific IgG, isotype control)

• For studying DHFR complexes, consider milder detergents (0.5% NP-40)

• Validate successful IP using Western blot with a different DHFR antibody clone

Antibodies specifically validated for immunoprecipitation, such as DHFR Antibody (A-9), will typically yield better results than those optimized solely for Western blotting .

What methods can resolve contradictory results obtained with different DHFR antibody clones?

When facing contradictory results from different DHFR antibody clones, implement this systematic troubleshooting approach:

Antibody characterization:

• Determine epitopes recognized by each antibody

• Assess specificity via Western blot in multiple cell lines (293T, Raji, C2C12, Rat-2)

• Verify performance in knockout/knockdown validation studies

• Review literature reports of each antibody's performance

Technical assessment:

• Standardize sample preparation across experiments

• Test multiple detection systems for each antibody

• Evaluate buffer compatibility with each antibody

• Consider effects of denaturing vs. native conditions on epitope accessibility

Biological considerations:

• Different antibodies may recognize distinct DHFR isoforms

• Post-translational modifications might affect epitope recognition

• DHFR-protein complexes could mask certain epitopes

Resolution strategy:

• Perform side-by-side comparison with standardized samples

• Correlate antibody detection with DHFR activity assays

• Consider using antibody combinations for comprehensive assessment

• When publishing, clearly report which epitopes/antibodies were used

This structured approach transforms contradictory results into deeper insights about DHFR biology and regulation.

How can DHFR antibodies be employed to study drug resistance mechanisms?

DHFR antibodies are valuable tools for investigating antifolate resistance mechanisms, particularly resistance to methotrexate (MTX):

Research applications:

• Quantification of DHFR expression levels:

  • Western blot analysis comparing resistant vs. sensitive cells

  • Immunohistochemistry in tumor samples

  • Flow cytometry for single-cell analysis

• Mutation-specific detection:

  • Development of antibodies targeting MTX-resistant DHFR variants

  • "Expression of methotrexate-resistant DHFR variants in normal hematopoietic cells represents a promising strategy to enhance methotrexate treatment efficacy"

• DHFR degradation kinetics:

  • Pulse-chase experiments with immunoprecipitation

  • Analysis of protein half-life in response to treatment

  • Monitoring degradation through inhibitors of NAD kinase that decrease NADPH levels

• DHFR-drug interaction studies:

  • Immunoprecipitation followed by drug binding assays

  • Antibody-based detection of conformational changes upon drug binding

Methodological framework:

• Establish resistant cell lines through gradual MTX exposure

• Compare DHFR expression using validated antibodies across multiple applications

• Correlate protein expression with enzymatic activity measurements

• Analyze cell cycle effects using propidium iodide staining and flow cytometry

• Validate findings in patient-derived samples

This comprehensive approach enables mechanistic understanding of DHFR-mediated drug resistance and facilitates development of strategies to overcome treatment limitations.

What are the optimal protocols for quantifying DHFR expression by immunohistochemistry?

Quantitative analysis of DHFR expression in immunohistochemistry requires standardized approaches:

Sample preparation:

• Use consistent fixation protocols (10% neutral buffered formalin for 24-48 hours)

• Perform heat-mediated antigen retrieval with Tris-EDTA buffer at pH 9.0

• For antibodies like ab124814, use a dilution of 1/1000 (0.20 μg/mL)

• Counterstain with hematoxylin for nuclear visualization

Scoring systems:

• H-score (combines intensity and percentage of positive cells)

• Allred score (useful for heterogeneous staining)

• Automated image analysis using pixel-based quantification

Analytical parameters:

• Staining intensity (negative, weak, moderate, strong)

• Percentage of positive cells

• Subcellular localization pattern

• Spatial distribution within tissue architecture

Standardization approach:

• Capture standardized digital images across all samples

• Apply consistent thresholding parameters

• Analyze multiple fields per sample (minimum 5-10 fields)

• Include internal control tissues in each batch

• Consider multi-observer scoring for validation

This systematic approach ensures reliable quantification and comparison of DHFR expression across different sample types and experimental conditions.

How can DHFR antibodies be used in multiplex immunofluorescence studies?

Multiplex immunofluorescence with DHFR antibodies allows simultaneous visualization of DHFR and other proteins:

Available conjugated antibodies:

• FITC-conjugated (sc-377091 FITC)

• PE-conjugated (sc-377091 PE)

• Alexa Fluor conjugates (sc-377091 AF488)

Multiplexing strategies:

• Sequential staining protocol:

  • Apply first primary antibody followed by fluorophore-conjugated secondary

  • Block remaining free binding sites

  • Apply DHFR antibody with differently-conjugated secondary antibody

  • Include nuclear counterstain (DAPI)

• Direct conjugate approach:

  • Use directly conjugated DHFR antibodies

  • Combine with other directly conjugated antibodies with non-overlapping spectra

  • Optimize concentrations to achieve balanced signal intensities

• Spectral unmixing:

  • Use specialized imaging systems capable of distinguishing overlapping fluorophores

  • Enable higher-order multiplexing (4+ targets simultaneously)

Optimization parameters:

• Antibody concentration and incubation time

• Order of antibody application

• Blocking between sequential staining steps

• Selection of compatible fluorophores with minimal spectral overlap

Controls for multiplex studies:

• Single-stained controls for spectral compensation

• Fluorescence-minus-one controls

• Absorption controls to verify no cross-reactivity between antibodies

This approach allows visualization of DHFR in relation to other pathway components or cellular structures in a spatial context.

What methodological approaches can assess DHFR protein-protein interactions?

DHFR interacts with various proteins and even binds its own mRNA . Several antibody-based approaches can investigate these interactions:

Co-immunoprecipitation (Co-IP):

• Use anti-DHFR antibodies to pull down DHFR and associated proteins

• Perform Western blot analysis of co-precipitated partners

• Consider mass spectrometry for unbiased identification of interactors

• Optimal lysis conditions: 150 mM NaCl, 5 mM EDTA, 50 mM HEPES (pH 7.5), 1.0% Triton X-100

Proximity Ligation Assay (PLA):

• Detect protein interactions in situ with <40 nm proximity

• Use anti-DHFR antibody with antibodies against suspected partners

• Visualize interactions as fluorescent spots via microscopy

• Quantify interaction frequency using automated image analysis

FRET/BRET approaches:

• Use fluorophore-conjugated DHFR antibodies (available as FITC, PE, Alexa Fluor conjugates)

• Pair with differently labeled antibodies against interaction partners

• Measure energy transfer as evidence of physical proximity

• Optimize donor:acceptor ratios for maximum sensitivity

ChIP and RIP approaches:

• Investigate DHFR interactions with nucleic acids

• Particularly useful for studying DHFR's role in binding its own mRNA

• Use highly specific DHFR antibodies validated for immunoprecipitation

These complementary approaches provide a comprehensive understanding of DHFR's protein interaction network in different cellular contexts.

How do different detection systems impact the sensitivity of DHFR Western blots?

Detection system selection significantly affects DHFR Western blot sensitivity and dynamic range:

Comparison of detection methods:

DHFR antibody conjugates available:

• HRP-conjugated (sc-377091 HRP) for direct chemiluminescent detection

• Various fluorophore conjugates for direct fluorescent detection

Optimization strategies:

• For chemiluminescence:

  • Use high-sensitivity substrates for low abundance detection

  • Optimize exposure times (multiple exposures recommended)

  • Consider digital imaging for better dynamic range

• For fluorescence:

  • Select appropriate wavelengths to minimize autofluorescence

  • Optimize scanning parameters (PMT gain, laser power)

  • Use compatible membrane types (low-fluorescence PVDF)

• General considerations:

  • Block thoroughly to minimize background

  • Include positive controls at different dilutions

  • Use purified recombinant DHFR as a standard curve

For DHFR detection, successful Western blot has been demonstrated using PVDF membrane probed with 1 μg/mL of Mouse Anti-Human DHFR Monoclonal Antibody followed by HRP-conjugated secondary antibody, visualizing DHFR at approximately 21 kDa .

What methodological approaches can address variability in DHFR antibody detection?

Variability in DHFR detection can stem from multiple sources that require systematic resolution:

Technical factors and solutions:

• Antibody lot-to-lot variation:

  • Create master antibody stocks for long-term studies

  • Validate each new lot against previous standards

  • Consider site-specific recombinant antibody technology

• Sample processing inconsistency:

  • Standardize lysis buffers and protein extraction protocols

  • Implement precise timing for all processing steps

  • Use automated systems where possible

• Detection system variability:

  • Calibrate imaging equipment regularly

  • Include standard curves on each blot

  • Use digital imaging for better reproducibility

Biological factors and controls:

• Cell cycle-dependent DHFR expression:

  • Synchronize cells when studying cell cycle-regulated proteins

  • Document cell confluence and passage number

  • Consider cell cycle analysis in parallel

• Growth conditions:

  • Standardize media composition and serum lots

  • Control temperature, CO₂, and humidity precisely

  • Document any deviations from standard conditions

Standardization approach:

• Implement detailed protocol documentation

• Use internal reference standards across experiments

• Include quantitative loading controls appropriate for each experiment

• Consider normalization to total protein methods

• Implement statistical approaches appropriate for the experimental design

By systematically addressing these variables, researchers can achieve more consistent and reliable DHFR detection across independent experiments.

How can DHFR antibodies be utilized to study the "DHFR handle" system for fusion protein applications?

The "DHFR handle" system offers unique applications for fusion protein studies. Based on research by Iwakura et al., this system can be applied immunologically for small peptides like leucine enkephalin (LEK) :

Principles of the DHFR handle system:

• DHFR serves as a protein fusion partner ("handle")

• Small peptides fused to DHFR maintain their conformational integrity

• The system enables large-scale preparation of otherwise difficult-to-produce peptides

• DHFR antibodies can be used to detect, purify, or track the fusion proteins

Experimental approach:

• Generate DHFR-peptide fusion constructs

• Express and purify using standard recombinant protein techniques

• Use anti-DHFR antibodies for:

  • Western blot detection of fusion proteins

  • Immunoprecipitation of fusion complexes

  • Immunoaffinity purification

  • Immunofluorescence localization studies

Validation methods:

• Competitive binding assays to assess epitope accessibility

• Verification that DHFR antibodies recognize the handle portion without interfering with peptide function

• IC₅₀ value determination for competitive binding (e.g., LEK showed IC₅₀ values of 3.74×10⁻⁶ and 4.66×10⁻⁶ M with two different monoclonal antibodies)

This approach is particularly valuable for studying small bioactive peptides and protein fragments that are otherwise challenging to work with due to size, stability, or detection limitations.

What considerations are important when studying DHFR expression across different cancer cell lines?

DHFR expression varies significantly across cancer cell lines, requiring careful experimental design:

Cell line selection considerations:

• Include both methotrexate-sensitive and resistant lines

• Select lines representing diverse tissue origins

• Consider lines with known DHFR gene amplification status

• Include normal cell counterparts for comparison

Validated cell lines for DHFR studies:

• 293T human embryonic kidney cells

• Raji human Burkitt's lymphoma cells

• MCF-7 human breast cancer cells

• C2C12 mouse myoblast cells

• Rat-2 rat embryonic fibroblast cells

Experimental standardization:

• Maintain consistent culture conditions across all lines

• Harvest cells at similar confluence (70-80%)

• Use multiple DHFR antibodies targeting different epitopes

• Correlate protein expression with mRNA levels and enzyme activity

Advanced analysis approaches:

• Compare subcellular localization patterns across cell types

• Assess response to folate pathway perturbations

• Correlate DHFR expression with proliferation rates and cell cycle profiles

• Consider drug sensitivity testing in relation to DHFR levels

By systematically characterizing DHFR expression across diverse cancer cell lines, researchers can gain insights into the relationship between DHFR expression, cell type specificity, and potential therapeutic targeting strategies.

How can researchers differentiate between DHFR isoforms using antibody-based approaches?

Differentiating between DHFR isoforms requires specialized antibody-based strategies:

Background on DHFR isoforms:

• Alternative splicing yields multiple DHFR isoforms

• The canonical human DHFR protein has 187 amino acid residues and a mass of 21.5 kDa

• DHFR can also bind its own mRNA and that of DHFR2

Antibody selection strategies:

• Isoform-specific antibodies:

  • Select antibodies targeting unique regions in specific isoforms

  • Validate specificity using recombinant isoform proteins

  • Consider custom antibody development for poorly characterized isoforms

• Differentiation by molecular weight:

  • Use high-resolution SDS-PAGE to separate closely related isoforms

  • Western blot with antibodies recognizing common epitopes

  • Compare to predicted molecular weights of known isoforms

• 2D gel electrophoresis approach:

  • Separate isoforms by both isoelectric point and molecular weight

  • Immunoblot to identify DHFR-specific spots

  • Perform mass spectrometry on isolated spots for definitive identification

• Immunoprecipitation with isoform discrimination:

  • Use isoform-specific antibodies for selective precipitation

  • Analyze precipitated proteins by mass spectrometry

  • Confirm differential interactions with binding partners

This methodological approach enables researchers to distinguish between closely related DHFR isoforms and study their specific functions in different cellular contexts.

What are the best approaches for correlating DHFR antibody staining with enzymatic activity?

Correlating DHFR antibody detection with enzymatic activity provides crucial validation:

Enzymatic activity measurement:

• The specific activity of DHFR can be determined through established protocols

• Typical assays monitor the conversion of dihydrofolate to tetrahydrofolate with NADPH as cofactor

• Spectrophotometric methods track NADPH oxidation at 340 nm

• Results can be expressed as nmol substrate converted per minute per mg protein

Correlation methodologies:

• Parallel analysis:

  • Process identical samples for both antibody detection and activity assays

  • Plot quantitative antibody signal versus specific activity

  • Calculate correlation coefficients and regression analysis

• Cell-based approaches:

  • Use cell lines with varying DHFR expression levels

  • Perform immunocytochemistry for spatial DHFR distribution

  • Correlate with enzymatic activity in cell lysates

  • Include MTX-resistant variants with altered DHFR properties

• Manipulation-based validation:

  • Perform DHFR knockdown/overexpression studies

  • Track changes in both antibody detection and enzyme activity

  • Include drug inhibition studies (e.g., methotrexate, NADPS)

• In situ approaches:

  • Develop histochemical assays for DHFR activity

  • Compare spatial patterns with immunohistochemistry results

  • Use dual labeling where feasible

This integrated approach validates antibody performance while providing insights into the relationship between DHFR protein levels and functional activity across different experimental conditions.

What emerging technologies are enhancing DHFR antibody applications in research?

Recent technological advances are expanding the utility of DHFR antibodies in research settings:

• Super-resolution microscopy:

  • Enables visualization of DHFR at the nanoscale level

  • Reveals previously undetectable subcellular distribution patterns

  • Requires highly specific antibodies with minimal background

• Mass cytometry (CyTOF):

  • Uses metal-conjugated antibodies for highly multiplexed cellular analysis

  • Allows simultaneous detection of DHFR with 40+ other proteins

  • Eliminates fluorescence spectrum limitations

• Proximity-dependent biotinylation:

  • DHFR-BioID or DHFR-APEX2 fusion proteins

  • Maps protein interaction networks in living cells

  • Can be detected with both anti-DHFR and anti-biotin antibodies

• Single-cell proteomics:

  • Measures DHFR levels in individual cells within heterogeneous populations

  • Correlates with other protein markers and cellular phenotypes

  • Requires highly sensitive and specific antibodies

• Antibody engineering:

  • Recombinant antibody technology for consistent performance

  • Site-specific conjugation for improved sensitivity

  • Nanobodies and single-chain antibodies for improved penetration

These emerging technologies are expanding the capabilities of DHFR antibody applications across multiple research domains, from basic biochemistry to translational medicine.

What are the most critical methodological considerations for publishing DHFR antibody-based research?

When publishing DHFR antibody-based research, address these critical methodological considerations:

• Comprehensive antibody reporting:

  • Manufacturer and catalog number

  • Clone identification (e.g., A-9, EPR5285, 872442)

  • Lot number and concentration used

  • Species and isotype (e.g., mouse monoclonal IgG2b κ)

  • Link to RRID (Research Resource Identifier) if available

• Validation documentation:

  • Specificity controls (knockout/knockdown, blocking peptide)

  • Cross-reactivity assessment for relevant species

  • Application-specific validation (not assuming WB validation transfers to IHC)

  • Lot-specific validation if using different lots

  • Inclusion of positive and negative controls

• Detailed methodological protocols:

  • Complete sample preparation procedures

  • Buffer compositions and reaction conditions

  • Incubation times and temperatures

  • Detection systems with complete parameters

  • Image acquisition settings and analysis parameters

• Quantification and statistics:

  • Clear description of quantification methods

  • Number of biological and technical replicates

  • Appropriate statistical tests with justification

  • Effect size reporting alongside p-values

  • Raw data availability statement