pof2 Antibody

What is the DIAPH2/POF2 protein and why is it significant in research?

DIAPH2 (diaphanous-related formin 2) is a 126 kDa cytoplasmic protein belonging to the diaphanous subfamily of actin-binding proteins. It plays a key role in actin cytoskeleton dynamics and cell migration. The protein is primarily localized in the cytoplasm, early endosomes, and cytosol . DIAPH2 has gained significant research interest due to its association with premature ovarian failure when the gene is disrupted . The X-chromosome gene DIAPH2 was found to be interrupted by a balanced X;12 translocation in a patient with POF who inherited the condition from her similarly affected mother .

What types of POF2/DIAPH2 antibodies are available for research?

Two main types of antibodies are available for DIAPH2/POF2 research:

Polyclonal Antibodies:

• Example: DIAPH2 Rabbit Polyclonal Antibody (CAB10209)

• Host: Rabbit

• Reactivity: Human, Mouse, Rat

• Applications: Western blot (WB), ELISA

• Immunogen: Recombinant fusion protein containing amino acids 1-120 of human DIAPH2

Monoclonal Antibodies:

• Example: Human DIAPH2 Monoclonal Antibody (MAB6806)

• Host: Mouse

• Reactivity: Human

• Applications: Western blot

• Immunogen: E. coli-derived recombinant human DIAPH2 (Val314-Gln540)

What are the recommended applications for POF2/DIAPH2 antibodies?

POF2/DIAPH2 antibodies have been validated for several research applications:

The antibodies can detect DIAPH2 in various sample types, including human tissues and cell lines such as SKOV3, SW620, NCI-H460, LO2, 293T, as well as mouse and rat tissues .

How can I optimize immunostaining protocols for POF2/DIAPH2 detection?

For optimal immunostaining with POF2/DIAPH2 antibodies, consider implementing these validated enhancement strategies:

Tissue Preparation:

• Use Davidson's solution (formaldehyde, absolute ethanol, glacial acetic acid, and water; 2:3:1:3) for fixation to preserve protein structure and epitope accessibility

• For paraffin-embedded sections, perform effective antigen retrieval using sodium citrate buffer (10 mM; pH 6.0) with microwave heating

• Block non-specific binding with PBS containing 3% normal horse serum (PBS-NHS)

Antibody Incubation Options:

• Two-Step Method: Primary antibody overnight at room temperature in PBS-NHS, followed by fluorochrome-conjugated secondary antibody for 30 minutes

• Three-Step Method for Enhanced Sensitivity: Primary antibody → biotinylated secondary antibody → streptavidin-conjugated fluorochrome

Signal Enhancement Techniques:

• Microwave irradiation: For frozen sections, perform three consecutive heat-cool cycles (5 min at 240W) in 1 mM EDTA (pH 8.0)

• Extended permeabilization with 1% Triton X-100 during primary antibody incubation

• Consider using the three-step detection method for weakly expressed targets

What are the challenges in detecting DIAPH2/POF2 in ovarian tissue samples?

Detecting DIAPH2/POF2 in ovarian tissues presents several challenges:

• Fixation-induced epitope masking: Ovarian tissue fixation can mask epitopes, requiring optimization of antigen retrieval methods

• Variable expression levels: DIAPH2 expression may vary across different cell types within the ovary and may be altered in pathological conditions

• Specificity concerns: Antibodies must distinguish between DIAPH2 and other members of the diaphanous protein family

• Detection in patient samples: POF patients may have mutations affecting epitope recognition, necessitating multiple antibodies targeting different regions

To address these challenges, researchers should:

• Include appropriate positive controls (e.g., tissues with known DIAPH2 expression)

• Use multiple antibodies recognizing different epitopes when possible

• Consider complementary detection methods to validate findings

How can POF2/DIAPH2 antibodies contribute to understanding premature ovarian failure pathogenesis?

POF2/DIAPH2 antibodies serve as valuable tools for investigating the molecular mechanisms underlying premature ovarian failure:

• Expression pattern analysis: Compare DIAPH2 expression patterns between normal ovarian tissues and those from POF patients

• Protein localization studies: Determine subcellular localization changes in POF-associated mutations

• Functional studies: Investigate how mutations affect DIAPH2's interaction with cytoskeletal components and RhoA signaling

• Genotype-phenotype correlations: Analyze how specific DIAPH2 mutations correlate with protein expression and POF phenotypes

A specific example of this application comes from research where:

• Site-directed mutagenesis was used to introduce POF-associated mutations (e.g., NM_006729.4:c.868A>G) into DIAPH2 expression constructs

• Antibodies were then employed to detect localization differences between wild-type and mutant proteins

• Constitutively active RhoA (RhoA-G14V) was co-expressed to study pathway interactions

What methodological approaches can be used to study the relationship between DIAPH2 mutations and ovarian function?

Researchers investigating DIAPH2's role in ovarian function can employ several methodological approaches:

• Immunohistochemical analysis of ovarian tissues:

  • Compare DIAPH2 expression patterns across different ovarian cell types

  • Examine changes in expression during follicular development

  • Analyze expression in POF patient samples versus controls

• Cell culture models:

  • Express wild-type and mutant DIAPH2 in relevant cell lines

  • Use antibodies to assess protein localization and interaction with cytoskeletal elements

  • Measure cellular phenotypes (e.g., membrane protrusions, cytoskeletal organization)

• Co-immunoprecipitation studies:

  • Identify protein-protein interactions affected by POF-associated mutations

  • Investigate DIAPH2 interactions with other proteins implicated in ovarian function

• Functional assays:

  • Assess cytoskeletal dynamics in cells expressing wild-type versus mutant DIAPH2

  • Measure membrane protrusion length and formation, which may be altered in POF-associated mutations

How can I validate the specificity of POF2/DIAPH2 antibodies?

Validating antibody specificity is crucial for reliable research results. For POF2/DIAPH2 antibodies, consider these validation approaches:

• Positive controls: Use samples known to express DIAPH2, such as:

  • Cell lines: SKOV3, SW620, NCI-H460, LO2, 293T

  • Tissues: Human testis, mouse liver, mouse kidney, rat liver

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight (approximately 125-130 kDa)

• Multiple antibody approach: Use antibodies targeting different epitopes to confirm results

  • Similar to the approach used in P2 protein studies where two monoclonal antibodies recognizing different epitopes provided complementary information

• Genetic knockdown/knockout validation: If possible, verify specificity by testing on samples with reduced or absent DIAPH2 expression

• Recombinant protein controls: Use purified recombinant DIAPH2 protein as a positive control for antibody binding specificity

How should I interpret contradictory results between different antibodies targeting POF2/DIAPH2?

When faced with contradictory results from different POF2/DIAPH2 antibodies, consider these factors:

• Epitope accessibility: Different antibodies target distinct epitopes that may be differentially accessible depending on:

  • Protein conformation

  • Post-translational modifications

  • Protein-protein interactions

  • Fixation conditions

• Isoform specificity: DIAPH2 has multiple isoforms (e.g., hDia2C) that may be recognized differently by various antibodies

• Technical considerations:

  • Differences in antibody affinity and avidity

  • Variations in experimental conditions

  • Sample preparation methods

For example, in studies of P2 protein antibodies, one antibody (2E6) recognized a surface-exposed determinant, while another (3F3) recognized an internal epitope, leading to different patterns of reactivity . Similar patterns may occur with DIAPH2/POF2 antibodies.

How can POF2/DIAPH2 antibodies be utilized in high-throughput screening approaches?

POF2/DIAPH2 antibodies can be adapted for high-throughput applications:

• Antibody microarrays:

  • Immobilize antibodies on arrays to detect DIAPH2 in multiple samples simultaneously

  • Use for screening patient cohorts for expression variations

• Flow cytometry-based screening:

  • Detect intracellular DIAPH2 in cell populations

  • Quantify expression levels across different cell types or experimental conditions

• Automated immunohistochemistry:

  • Process multiple tissue samples simultaneously

  • Quantify expression patterns across tissue microarrays

These approaches can be modeled after those used in other antibody studies, such as the high-throughput screening methods developed for antibody profiling in SARS-CoV-2 research .

What emerging technologies might enhance the utility of POF2/DIAPH2 antibodies in research?

Several emerging technologies could enhance POF2/DIAPH2 antibody applications:

• Biophysics-informed modeling:

  • Computational approaches to predict antibody binding properties

  • Design of antibodies with enhanced specificity for particular DIAPH2 domains

• Proximity labeling techniques:

  • Antibody-based proximity labeling to identify DIAPH2 interaction partners

  • Map protein interaction networks in normal versus POF conditions

• Single-cell antibody-based techniques:

  • Analyze DIAPH2 expression at the single-cell level within heterogeneous ovarian tissues

  • Correlate with cellular phenotypes and developmental stages

• In vivo imaging:

  • Develop fluorescently labeled antibody fragments for live-cell imaging

  • Track DIAPH2 dynamics in cellular models

These approaches build upon established methodologies while incorporating technological advances to gain deeper insights into DIAPH2 function and its role in premature ovarian failure.