ONECUT2 Antibody

What is ONECUT2 and why is it significant in research?

ONECUT2 is a monomeric 54 kDa transcription factor belonging to the One Cut-domain containing class of homeodomain proteins. Human ONECUT2 is 504 amino acids in length and contains two DNA binding regions: a CUT domain (aa 331-410) and a homeobox domain (aa 427-481) . It functions as a transcriptional regulator impacting the expression of genes such as Ngn3, OPN, and Thbs4 . ONECUT2 is significant in research due to its role in embryonic development, cell differentiation, and its implication in various diseases, particularly in cancer progression where it acts as a master regulator of lineage plasticity .

Which ONECUT2 antibody formats are available for different applications?

ONECUT2 antibodies are available as polyclonal and monoclonal formats from multiple vendors, with various host species including rabbit and sheep. Most commonly available antibodies are unconjugated, though some are available with fluorescent conjugates for direct detection . For optimal experimental outcomes, researchers should select antibodies validated for their specific application:

How can I validate ONECUT2 antibody specificity?

Validating antibody specificity is critical for ensuring reliable results. A multi-faceted approach is recommended:

• Analyze expression in known positive controls (HepG2, HeLa, HEK-293 cells)

• Include negative controls lacking ONECUT2 expression

• Perform ONECUT2 knockdown/knockout experiments to confirm signal reduction

• Verify expected molecular weight (54-60 kDa) in Western blot applications

• Confirm expected nuclear localization in immunostaining applications

• Use multiple antibodies targeting different epitopes for confirmation

• Check for cross-reactivity with other ONECUT family members (ONECUT1, ONECUT3)

What are the optimal conditions for immunostaining with ONECUT2 antibodies?

For successful immunostaining with ONECUT2 antibodies:

• Fixation: Standard fixation protocols with formaldehyde or paraformaldehyde are generally effective

• Antigen retrieval: Use TE buffer pH 9.0 as primary choice; citrate buffer pH 6.0 can be used as an alternative

• Blocking: Standard blocking with serum or BSA (typically 5-10%)

• Primary antibody: Dilute 1:200-1:800 for IHC or 0.25-2 μg/mL for ICC/IF

• Incubation: Typically 1-3 hours at room temperature or overnight at 4°C

• Detection system: For immunofluorescence, secondary antibodies such as NorthernLights 557-conjugated Anti-Sheep IgG have been successfully used

• Counterstaining: DAPI works well for nuclear counterstaining as ONECUT2 shows specific localization to nuclei

How should ONECUT2 antibodies be stored and handled to maintain activity?

Proper storage and handling of ONECUT2 antibodies are essential for maintaining their activity:

• Long-term storage: Store at -20°C in aliquots to minimize freeze-thaw cycles

• Short-term storage: 4°C is acceptable for short periods (up to one week)

• Buffer composition: Most ONECUT2 antibodies are supplied in PBS with 0.02% sodium azide and 40-50% glycerol at pH 7.2-7.3

• Stability: Many ONECUT2 antibodies remain stable for one year after shipment when properly stored

• Aliquoting: While some formulations may not require aliquoting for -20°C storage, it's generally recommended to minimize freeze-thaw cycles

• Working dilutions: Prepare fresh working dilutions on the day of use

What cell models are most appropriate for studying ONECUT2?

Based on validated expression patterns, the following cell models are appropriate for ONECUT2 research:

How can ONECUT2 antibodies be utilized to study treatment resistance in prostate cancer?

ONECUT2 has emerged as a critical factor in treatment resistance, particularly in prostate cancer where it is active in approximately 60% of metastatic castration-resistant cases . Researchers can employ ONECUT2 antibodies to:

• Perform ChIP-seq to identify ONECUT2 binding sites and target genes in resistant versus sensitive cells

• Evaluate ONECUT2 expression changes before and after treatment with androgen receptor signaling inhibitors (ARSIs)

• Analyze co-localization with other resistance-associated factors

• Monitor ONECUT2 expression in response to targeted inhibitors

• Correlate ONECUT2 nuclear localization with treatment outcomes in patient samples

Research has demonstrated that ONECUT2 overexpression alone resulted in enzalutamide-resistant phenotypes in LNCaP and LAPC4 prostate cancer models by activating multiple AR-indifferent lineages . This makes ONECUT2 a valuable marker for monitoring therapy resistance development.

What is the significance of ONECUT2 in driving lineage plasticity in cancer?

ONECUT2 functions as a master regulator of lineage plasticity in cancer, particularly in contexts requiring adaptation to therapeutic pressure:

• It activates diverse resistance drivers associated with adenocarcinoma, stem-like, and neuroendocrine variants

• It operates globally at the chromatin level to activate numerous AR-indifferent, lineage-defining factors

• It induces treatment-emergent lineage variation in prostate cancer

• It promotes resistance through multiple mechanisms associated with different cancer cell phenotypes

Using ONECUT2 antibodies in combination with lineage markers can help researchers track cellular reprogramming events during disease progression and therapeutic resistance development. ChIP studies using validated ONECUT2 antibodies can identify direct targets responsible for phenotypic transitions.

How does ONECUT2 function in intestinal epithelial differentiation?

Recent multi-omics research has revealed a critical role for ONECUT2 in intestinal epithelial differentiation:

• ONECUT2 acts downstream of the RANK/RANKL signaling axis in the small intestine

• It supports enterocyte differentiation while restricting microfold (M) cell lineage specification

• It influences cell fate balance in Peyer's patches, impacting gut-associated immune responses

• Its expression and activity can be modulated experimentally in intestinal organoid models

Using ONECUT2 antibodies in combination with intestinal lineage markers enables tracking of differentiation trajectories and cell fate decisions in both normal and pathological conditions. ChIP-seq with ONECUT2 antibodies can identify the regulatory elements and target genes controlling these differentiation processes.

What protocols are recommended for ChIP experiments with ONECUT2 antibodies?

For optimal Chromatin Immunoprecipitation (ChIP) with ONECUT2 antibodies:

• Chromatin preparation: Fix cells with 1% formaldehyde for 10 minutes at room temperature

• Sonication: Fragment chromatin to 200-500 bp

• Pre-clearing: Save 1% of input material before immunoprecipitation

• Immunoprecipitation: Incubate chromatin with 1 μg of anti-ONECUT2 antibody in appropriate dilution buffer (1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8, 167 mM NaCl, with protease inhibitors)

• Incubation: Rotate overnight at 4°C

• Purification: Use protein A and protein G Dynabeads to purify antibody-bound chromatin

• Washing: Perform sequential washes with increasing NaCl concentration followed by TE buffer

• Elution and reverse crosslinking: Standard protocols apply

• DNA purification: Standard column-based methods

• Validation: qPCR for known ONECUT2 targets or sequencing

What are common issues with Western blotting using ONECUT2 antibodies and how can they be resolved?

When performing Western blots with ONECUT2 antibodies, researchers may encounter several issues:

How can inconsistent immunostaining results with ONECUT2 antibodies be addressed?

For consistent immunostaining results:

• Fixation optimization: Test different fixation protocols (duration, temperature, fixative concentration)

• Antigen retrieval comparison: Compare TE buffer pH 9.0 versus citrate buffer pH 6.0

• Blocking optimization: Test different blocking reagents (normal serum, BSA, commercial blockers)

• Antibody titration: Perform careful antibody dilution series to determine optimal concentration

• Incubation conditions: Compare room temperature versus 4°C incubation

• Detection system selection: For fluorescence detection, NorthernLights 557-conjugated secondary antibodies have proven effective

• Counterstaining protocol: DAPI is effective for visualizing ONECUT2 nuclear localization

How can ONECUT2 antibodies contribute to understanding transcriptional networks in development and disease?

ONECUT2 antibodies are valuable tools for deciphering complex transcriptional networks:

• ChIP-seq applications can identify genome-wide binding sites and co-factors

• Sequential ChIP (Re-ChIP) can determine co-occupancy with other transcription factors

• CUT&RUN/CUT&Tag provide higher resolution alternatives to traditional ChIP

• Proximity ligation assays can detect protein-protein interactions in situ

• Combining with single-cell approaches can reveal heterogeneity in ONECUT2 activity

• Integration with epigenomic data can uncover relationships between ONECUT2 binding and chromatin state

Recent multi-omics approaches have successfully integrated chromatin accessibility profiling with transcription factor dynamics and single-cell RNA sequencing to understand ONECUT2's role in intestinal epithelial differentiation .

What recent therapeutic approaches target ONECUT2?

Recent research has identified ONECUT2 as a potential therapeutic target:

• Small molecule inhibitors targeting ONECUT2 have shown promise in suppressing established prostate cancer metastases in mice

• ONECUT2 inhibition may be particularly effective against treatment-resistant cancer variants

• Combination approaches targeting ONECUT2 alongside standard therapies may prevent resistance development

• Biomarkers associated with ONECUT2 activity could help identify patients likely to benefit from targeted therapies

• Understanding ONECUT2's normal physiological roles is essential for predicting potential side effects of therapeutic targeting