DHFR Antibody

What is DHFR and why is it an important research target?

Dihydrofolate reductase (DHFR) is a key enzyme in folate metabolism that contributes to the de novo mitochondrial thymidylate biosynthesis pathway. It catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis . DHFR is an established anti-cancer drug target whose inhibition disrupts folate metabolism and STAT3-dependent gene expression . As a ubiquitously expressed monomer considered to be a housekeeping gene, DHFR participates in multiple pathways across different cell types, making it a valuable target for both basic research and therapeutic development .

What are the structural and functional characteristics of human DHFR?

Human DHFR is a 21-23 kDa protein consisting of 187 amino acid residues with one DHFR domain (amino acids 4-185) . It exists in two pools within cells: one containing DHFR bound to its own RNA where it acts as a transcriptional repressor, and another containing DHFR bound to NADPH . The protein is localized in both mitochondria and cytoplasm . Its mRNA binding motif is suggested to involve Cys6, Leu22, Glu30, and Ser118, which can affect regulatory mechanisms .

What are the different types of DHFR antibodies available for research?

Research-grade DHFR antibodies are available in several formats:

Different antibodies target specific regions of DHFR, including the N-terminus (amino acids 1-50) or regions surrounding particular residues such as Gly175 .

How should DHFR antibodies be optimized for Western blotting applications?

For optimal Western blot results with DHFR antibodies:

• Sample preparation: Prepare whole cell lysates from established cell lines known to express DHFR (HeLa, Jurkat, COLO 205, HEK293T)

• Loading amount: Load 20-30 μg of protein lysate per lane for adequate detection of endogenous DHFR

• Blocking conditions: Use 5% non-fat dry milk in TBST as an optimal blocking buffer

• Antibody dilutions: Dilutions range from 1:1000 to 1:10,000 depending on the specific antibody; monoclonal antibodies often perform well at higher dilutions (1:10,000)

• Expected band size: Look for a specific band at approximately 21-22 kDa

• Controls: Include positive controls such as recombinant DHFR protein at 0.1-0.2 μg

The experimental data shows that DHFR antibodies can detect the protein across multiple species including human, mouse, and rat samples due to high sequence homology (90% amino acid sequence identity) .

What are the optimal conditions for DHFR immunohistochemistry and immunocytochemistry?

For successful IHC and ICC experiments:

Immunohistochemistry (IHC-P):

• Antigen retrieval: Heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

• Blocking: 10% goat serum is recommended to reduce background

• Antibody concentration: 2 μg/ml for paraffin-embedded sections

• Incubation conditions: Overnight at 4°C for primary antibody

• Detection systems: HRP-conjugated secondary antibodies with DAB chromogen work well

• Validated tissues: Colorectal cancer tissue, small intestine, and colon show strong DHFR expression

Immunocytochemistry/Immunofluorescence (ICC/IF):

• Cell fixation: Paraformaldehyde (4%) for cell fixation and permeabilization

• Antibody concentration: 5-10 μg/ml for cellular staining

• Incubation time: 3 hours at room temperature

• Visualization: Use appropriate fluorophore-conjugated secondary antibodies (e.g., DyLight®550, NorthernLights™ 557)

• Counterstaining: DAPI for nuclear visualization

• Validated cell lines: U20S, MCF-7, PC-3 cells show reliable DHFR staining patterns

How can DHFR target engagement be measured in cells?

Modern techniques for measuring DHFR target engagement include:

• Thermal Proteome Profiling (TPP): This technique helps confirm DHFR inhibitor binding in cellular contexts and can identify additional targets for inhibitors like methotrexate

• DHFR accumulation assay: Measuring DHFR protein accumulation following inhibitor treatment serves as an indicator of target engagement

• Biochemical assays coupled with cell-based assays: This combination provides a more comprehensive assessment of compound efficacy:

  • Biochemical assays evaluate direct inhibitory activity

  • Cell-based assays demonstrate target engagement in the cellular environment

Research has shown that compounds like Cycloguanil and its analogues can engage DHFR in cells at sub-nanomolar concentrations, although growth impairments may not be observed until higher concentrations are reached .

How should researchers validate the specificity of DHFR antibodies?

Comprehensive validation should include:

• Multiple detection methods: Cross-validate results using different techniques (WB, IHC, IF)

• Species cross-reactivity testing: Validate across human, mouse, and rat samples due to the 90% sequence homology

• Control experiments:

  • Positive controls: Use recombinant DHFR protein

  • Tissue-specific expression verification: Compare expression levels in tissues known to express DHFR (liver, kidney, colorectal tissue)

  • Cell line panels: Test across multiple cell lines (Jurkat, U937, C6, RAW 264.7, PC-12)

• Knockout/knockdown validation: Compare antibody signal in DHFR-knockout or knockdown samples (like DG44 cells that lack DHFR)

• Blocking peptide competition: Use the immunizing peptide to confirm signal specificity

Western blot data should show a clear band at the expected molecular weight of 21-22 kDa without significant non-specific banding .

What are common pitfalls in DHFR antibody-based experiments and how can they be avoided?

Common challenges and solutions include:

• Non-specific binding:

  • Problem: Background staining in IHC/IF or multiple bands in WB

  • Solution: Optimize blocking (5% NFDM/TBST for WB, 10% goat serum for IHC/IF) and increase antibody dilution

• Epitope masking:

  • Problem: Reduced or absent signal due to protein-protein interactions

  • Solution: Use multiple antibodies targeting different epitopes; some target the N-terminus (aa 1-50) while others target regions near Gly175

• Variable expression levels:

  • Problem: DHFR expression can vary across tissues and cell types

  • Solution: Include positive control samples with known DHFR expression (Jurkat, HEK293T)

• Species cross-reactivity issues:

  • Problem: Unexpected cross-reactivity or lack of expected cross-reactivity

  • Solution: Choose antibodies validated across species of interest; human DHFR shares 90% sequence identity with mouse and rat DHFR

• Signal detection sensitivity:

  • Problem: Weak signal despite adequate protein expression

  • Solution: Use signal enhancement systems; for IHC, employ HRP Conjugated Super Vision Assay Kit with DAB as chromogen

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

DHFR antibodies are valuable tools for investigating drug resistance mechanisms:

• Protein expression analysis: Use Western blotting to quantify DHFR overexpression, which is directly associated with methotrexate (MTX) resistance. Studies have shown that cells with increased DHFR protein levels (5-fold higher) demonstrate significantly higher MTX resistance (IC₅₀ values of 1.03 μM vs 0.29 μM in wild-type cells)

• SNP effects on DHFR expression: Investigate how SNPs in the DHFR gene affect antibody binding and protein levels. The 829C→T SNP near the miR-24 binding site in the 3′ UTR stabilizes DHFR mRNA, leading to 19-fold higher mRNA levels and 5-fold higher protein levels

• miRNA regulation studies: Examine how miRNAs like miR-24 regulate DHFR expression by binding to the 3′ UTR. Combine with DHFR antibodies to assess protein levels after miRNA overexpression or inhibition

• Target engagement analysis: Use DHFR antibodies in thermal shift assays to evaluate how mutations affect drug binding. This approach helps determine if resistance is due to reduced target binding or post-binding mechanisms

• Subcellular localization changes: Investigate whether drug resistance correlates with altered subcellular distribution of DHFR between cytoplasmic and mitochondrial compartments

What are the latest approaches for using DHFR antibodies in cancer research?

Cutting-edge applications include:

• Companion diagnostics development: DHFR antibodies can be used to stratify patients based on DHFR expression levels, potentially predicting responsiveness to antifolate therapies

• Polypharmacological studies: Recent research has illuminated that some DHFR inhibitors have additional targets. DHFR antibodies can help distinguish between on-target and off-target effects through target engagement assays

• DHFR inhibitor screening:

  • Computational modeling identifies promising inhibitor candidates

  • NCI-60 Human Tumor Cell Line Screening data provides efficacy information

  • DHFR antibodies confirm target engagement in cellular contexts

• Compensatory pathway investigation: Study how cancer cells adapt to DHFR inhibition by examining expression changes in related enzymes. For example, examining the relationship between DHFR inhibition and thymidylate synthase activity

• Novel therapeutic strategy development: Recent work has shown that the antimalarial drug Pyrimethamine selectively inhibits human DHFR in cancer cell lines, which is linked to downstream inhibition of STAT3 signaling. DHFR antibodies are crucial for confirming the mechanism of action

How can DHFR antibodies be integrated with other molecular biology techniques for comprehensive studies?

Integrative approaches include:

• CRISPR/Cas9 gene editing combined with antibody-based detection:

  • Generate DHFR knockout or point mutation cell lines

  • Use DHFR antibodies to confirm knockout efficiency or study mutant protein expression

  • Assess functional consequences on folate metabolism and cell growth

• Proteomics and interactome studies:

  • Use DHFR antibodies for immunoprecipitation followed by mass spectrometry

  • Identify novel DHFR-interacting proteins under different conditions

  • Map the dynamic DHFR interactome in response to drug treatment

• Patient-derived xenograft (PDX) models:

  • Use DHFR antibodies to characterize DHFR expression in PDX tumors

  • Correlate expression with drug response

  • Develop personalized treatment strategies based on DHFR status

• Single-cell analysis techniques:

  • Combine flow cytometry with DHFR antibodies to quantify heterogeneity

  • Link DHFR expression levels to cell cycle phases or differentiation states

  • Identify resistant subpopulations within tumors

• Computational biology integration:

  • Use antibody-derived expression data to validate in silico predictions

  • Incorporate DHFR expression data into systems biology models

  • Predict synergistic drug combinations targeting DHFR and complementary pathways

How are DHFR antibodies being used to study the relationship between DHFR and microRNAs?

Recent studies have revealed important interactions between DHFR and microRNAs:

• miR-24 regulatory mechanism: DHFR antibodies have been instrumental in confirming that miR-24 regulates DHFR expression. When miR-24 binds to the 3′ UTR of DHFR mRNA, it leads to downregulation of DHFR protein. This regulation can be disrupted by SNPs like 829C→T, which interferes with miR-24 binding

• mRNA half-life assessment: Studies using DHFR antibodies have shown that the 829C→T SNP increases DHFR mRNA half-life by 2-fold, contributing to protein overexpression and drug resistance

• Therapeutic potential of miRNA mimics: Research suggests that miR-24 mimics may be valuable alone or in combination with methotrexate in treatment, with DHFR antibodies being crucial for monitoring the effectiveness of such approaches

• SNP screening and personalized medicine: DHFR antibodies can help assess how various naturally occurring miRSNPs (SNPs located at or near microRNA binding sites) affect DHFR expression across different ethnic groups, potentially informing treatment outcomes and toxicity profiles

• Combination therapy development: Understanding miRNA-mediated regulation of DHFR using antibody-based detection methods can lead to novel combination therapies targeting both the enzyme and its regulatory mechanisms

What methodological considerations are important when using DHFR antibodies for flow cytometry?

For optimal flow cytometry results with DHFR antibodies:

• Cell preparation:

  • Fix cells with 4% paraformaldehyde to maintain cellular integrity

  • Permeabilize with appropriate buffer to allow antibody access to intracellular DHFR

• Blocking conditions:

  • Block with 10% normal goat serum to reduce non-specific binding

  • Use species-matched serum to the secondary antibody host

• Antibody concentration and incubation:

  • Use 1 μg/1×10⁶ cells for primary antibody

  • Incubate for 30 minutes at 20°C for optimal binding

• Controls:

  • Include isotype control antibody (e.g., rabbit IgG at 1 μg/1×10⁶ cells)

  • Use unlabelled sample without primary and secondary antibody as blank control

  • Include positive control cell lines with known DHFR expression (PC-3, Jurkat)

• Secondary antibody selection:

  • Use fluorophore-conjugated secondary antibodies (e.g., DyLight®488)

  • Maintain consistent 5-10 μg/1×10⁶ cells concentration

  • Incubate for 30 minutes at 20°C

• Data analysis considerations:

  • Analyze shifts in fluorescence intensity to quantify DHFR expression

  • Compare with standard cell lines to normalize expression levels

  • Account for autofluorescence when selecting fluorophores