ASPHD2 Antibody

What is ASPHD2 protein and why is it a significant research target?

ASPHD2 (Aspartate beta-Hydroxylase Domain Containing 2) functions as a histone-binding protein with crucial roles in chromatin organization and epigenetic regulation mechanisms . The protein is predicted to enable dioxygenase activity and metal ion binding, and is involved in peptidyl-amino acid modification processes . Its significance as a research target stems from its involvement in gene silencing and transcriptional regulation, which underscores its importance in cancer progression and growth . ASPHD2's roles in these fundamental cellular processes make it a promising target for cancer therapy and drug development research .

What applications are ASPHD2 antibodies commonly used for?

ASPHD2 antibodies are primarily validated for use in ELISA and Western Blotting (WB) applications . For ELISA applications, the recommended dilution ranges from 1:2000 to 1:10000, while for Western Blotting, the suggested dilution range is 1:500 to 1:2000 . Some ASPHD2 antibodies may also be suitable for immunofluorescence and immunohistochemistry applications, enabling precise detection and analysis of this protein in different cell types . The versatility of these applications allows researchers to investigate ASPHD2's function in cancer biology and gene regulation mechanisms.

What are the characteristics of commercially available ASPHD2 antibodies?

Most commercially available ASPHD2 antibodies are polyclonal antibodies raised in rabbits with high specificity for human samples . They typically recognize specific amino acid sequences within the ASPHD2 protein, such as amino acids 80-369, 165-194, or 107-156 . These antibodies are available in various forms, including:

These antibodies demonstrate high purity levels (>95%) through Protein G purification methods .

How should I optimize Western blot conditions for ASPHD2 antibody detection?

When optimizing Western blot conditions for ASPHD2 antibody detection, consider the following methodological approach:

• Sample preparation: Use whole cell lysates (such as from HeLa or HL60 cell lines) that are known to express ASPHD2 .

• Antibody concentration: Start with a concentration of 4μg/ml of the ASPHD2 antibody, as this has been validated in previous experiments .

• Secondary antibody: Use goat polyclonal anti-rabbit IgG at 1/10000 dilution for optimal signal detection .

• Band identification: The predicted molecular weight of ASPHD2 is 42 kDa, which should align with the observed band size in your Western blot .

• Blocking conditions: Use 5% non-fat dry milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature to minimize non-specific binding.

• Incubation times: Primary antibody incubation should be performed overnight at 4°C, while secondary antibody incubation should be limited to 1 hour at room temperature.

• Washing steps: Implement stringent washing steps (at least 3-5 washes of 5-10 minutes each) with TBST to reduce background signal.

What controls should I include when using ASPHD2 antibodies in my experiments?

Incorporating appropriate controls is crucial for validating results with ASPHD2 antibodies:

• Positive control: Include samples from cell lines known to express ASPHD2, such as HeLa or HL60 whole cell lysates .

• Negative control: Use samples from cell lines or tissues with low or no expression of ASPHD2, or use genetic knockdown/knockout models.

• Isotype control: Include a non-specific antibody of the same isotype (rabbit IgG) to assess non-specific binding.

• Loading control: For Western blots, include detection of housekeeping proteins (e.g., GAPDH, β-actin) to ensure equal loading across lanes.

• Peptide competition: Preincubate the antibody with the immunizing peptide to confirm specificity.

• Secondary antibody-only control: Omit the primary antibody to assess background from the secondary antibody.

• Dilution series: Run a dilution series of your positive control to establish the linear range of detection.

How can ASPHD2 antibodies be used to investigate chromatin organization and epigenetic regulation?

ASPHD2 antibodies can be leveraged for advanced investigation of chromatin organization and epigenetic regulation through several sophisticated approaches:

• Chromatin Immunoprecipitation (ChIP): Use ASPHD2 antibodies to immunoprecipitate ASPHD2-bound chromatin regions, followed by sequencing (ChIP-seq) to identify genome-wide binding patterns and association with specific histone modifications.

• Co-immunoprecipitation (Co-IP): Apply ASPHD2 antibodies to pull down ASPHD2 protein complexes, followed by mass spectrometry to identify interaction partners involved in chromatin remodeling and epigenetic processes.

• Proximity Ligation Assay (PLA): Combine ASPHD2 antibodies with antibodies against histone modifications or other chromatin-associated proteins to visualize and quantify their co-localization and potential interactions in situ.

• FRAP (Fluorescence Recovery After Photobleaching): Use fluorescently labeled ASPHD2 antibodies to study the dynamics of ASPHD2 binding to chromatin in living cells.

• Super-resolution microscopy: Apply FITC-conjugated ASPHD2 antibodies to investigate the spatial organization of ASPHD2 in relation to chromatin domains with nanometer precision.

These methodologies can provide crucial insights into ASPHD2's role in gene silencing and transcriptional regulation mechanisms involved in cancer progression.

What are the considerations for using ASPHD2 antibodies in cancer research models?

When using ASPHD2 antibodies in cancer research models, researchers should consider the following methodological aspects:

• Cancer type selection: Given ASPHD2's role in gene regulation and cancer progression , select cancer models where epigenetic dysregulation is well-documented (e.g., breast, colon, or lung cancer models).

• Expression profiling: Before antibody application, validate ASPHD2 expression levels across different cancer cell lines using qRT-PCR to identify high and low expressors.

• Clinical sample validation: When transitioning to patient-derived samples, validate antibody performance on tissue microarrays containing multiple cancer types and stages.

• Therapeutic response monitoring: Use ASPHD2 antibodies to monitor changes in expression or localization following treatment with epigenetic modulators or other cancer therapeutics.

• Cross-species considerations: When using animal models, verify antibody cross-reactivity with the species of interest, as some ASPHD2 antibodies react with multiple species including mouse, rat, and other mammals .

• Multiplexed analysis: Combine ASPHD2 antibody staining with markers of cell proliferation, apoptosis, or stemness to correlate ASPHD2 status with cancer phenotypes.

• 3D culture systems: Validate antibody penetration and performance in 3D culture models that better recapitulate tumor architecture compared to monolayer cultures.

What are common issues with ASPHD2 antibody specificity and how can they be addressed?

Researchers may encounter several specificity issues when working with ASPHD2 antibodies, which can be addressed through these methodological approaches:

• Multiple bands in Western blotting: If observing bands at sizes other than the expected 42 kDa , consider:

  • Optimizing sample preparation protocols to reduce protein degradation

  • Using fresher antibody aliquots to minimize degradation

  • Implementing more stringent blocking conditions (5-10% BSA or milk)

  • Testing antibodies targeting different epitopes of ASPHD2 (e.g., AA 80-369 vs. AA 165-194)

• Cross-reactivity with related proteins: ASPHD2 belongs to a family of proteins with aspartate beta-hydroxylase domains, which may cause cross-reactivity. Address this by:

  • Performing immunoprecipitation followed by mass spectrometry to confirm antibody targets

  • Validating results with multiple antibodies targeting different epitopes

  • Using ASPHD2 knockout/knockdown controls to confirm specificity

• Background in immunostaining: Minimize by:

  • Testing a range of antibody dilutions (starting with 1:500-1:2000 for WB)

  • Implementing more extensive washing steps

  • Using alternative blocking reagents (e.g., fish gelatin instead of BSA)

  • Optimizing fixation conditions for the specific tissue or cell type

How can I validate the functional relevance of ASPHD2 detection in my experimental system?

To validate that your ASPHD2 antibody detection is functionally relevant to your research question:

• Gene modulation experiments: Correlate antibody signal with ASPHD2 expression in:

  • siRNA or shRNA knockdown experiments

  • CRISPR-Cas9 knockout models

  • Overexpression systems using ASPHD2 expression vectors

• Functional assays: Link ASPHD2 detection to functional outcomes by:

  • Assessing changes in gene expression profiles following ASPHD2 modulation

  • Measuring cell proliferation, migration, or invasion in relation to ASPHD2 levels

  • Evaluating chromatin accessibility using ATAC-seq in systems with varying ASPHD2 levels

• Pharmacological validation: Use inhibitors of pathways related to ASPHD2 function and monitor changes in:

  • ASPHD2 protein levels

  • ASPHD2 subcellular localization

  • Post-translational modifications of ASPHD2

• Disease model correlation: Correlate ASPHD2 antibody signals with:

  • Disease progression in animal models

  • Patient clinical outcomes in tissue microarray studies

  • Response to relevant therapeutics in preclinical models

How can conjugated ASPHD2 antibodies enhance multiparameter analysis in cancer research?

Conjugated ASPHD2 antibodies offer powerful capabilities for multiparameter analysis in cancer research through these methodological approaches:

• Flow cytometry applications: FITC- or APC-conjugated ASPHD2 antibodies enable:

  • Simultaneous analysis of ASPHD2 with cell surface markers to identify specific cell populations

  • Correlation of ASPHD2 expression with cell cycle status using DNA content dyes

  • Assessment of ASPHD2 in relation to stemness markers in cancer stem cell populations

• Multiplex immunofluorescence imaging: Various conjugated forms of ASPHD2 antibodies allow:

  • Co-localization studies with multiple markers in the same tissue section

  • Spatial relationship analysis between ASPHD2 and the tumor microenvironment

  • Quantitative image analysis of heterogeneous ASPHD2 expression in tumors

• Mass cytometry (CyTOF): Metal-conjugated ASPHD2 antibodies enable:

  • High-dimensional analysis (30+ parameters) of ASPHD2 in relation to numerous cellular markers

  • Detailed phenotyping of rare cell populations expressing ASPHD2

  • Unbiased clustering of cells based on ASPHD2 and multiple other markers

• Single-cell technologies: Biotin-conjugated ASPHD2 antibodies facilitate:

  • Integration with single-cell RNA-seq protocols to correlate protein and mRNA levels

  • Expansion of CITE-seq approaches to include ASPHD2 in cellular phenotyping

  • Spatial transcriptomics applications linking ASPHD2 protein localization with local gene expression

What emerging technologies could enhance ASPHD2 antibody-based research?

Several cutting-edge technologies are poised to advance ASPHD2 antibody-based research:

• Nanobody and recombinant antibody approaches:

  • Development of smaller ASPHD2-targeting molecules with improved tissue penetration

  • Creation of bispecific antibodies targeting ASPHD2 and other epigenetic regulators simultaneously

  • Engineering of intrabodies for live-cell tracking of ASPHD2 dynamics

• Advanced imaging technologies:

  • Lattice light-sheet microscopy for real-time tracking of ASPHD2 in living cells

  • Expansion microscopy to resolve ASPHD2's precise localization within chromatin structures

  • Correlative light and electron microscopy to link ASPHD2 function to ultrastructural features

• Functional screens:

  • CRISPR activation/inhibition screens combined with ASPHD2 antibody-based readouts

  • Antibody-enabled spatial proteomics to map ASPHD2's interaction landscape

  • Optogenetic approaches combined with ASPHD2 antibody detection to study dynamic responses

• Translational applications:

  • Development of ASPHD2-targeted antibody-drug conjugates for therapeutic applications

  • Companion diagnostic approaches using ASPHD2 antibodies to stratify patients for targeted therapies

  • Liquid biopsy applications detecting ASPHD2 or its fragments in circulation

How might ASPHD2 antibody research evolve in relation to precision medicine approaches?

The future of ASPHD2 antibody research in precision medicine likely encompasses several exciting directions:

• Biomarker development: ASPHD2 antibodies may evolve into critical tools for patient stratification by:

  • Identifying tumors dependent on ASPHD2-mediated epigenetic regulation

  • Predicting response to epigenetic therapies based on ASPHD2 expression patterns

  • Monitoring treatment efficacy through quantitative assessment of ASPHD2 levels or localization

• Therapeutic targeting: Beyond research applications, ASPHD2 antibodies might be developed into therapeutic agents through:

  • Antibody-drug conjugates specifically delivering cytotoxic agents to ASPHD2-expressing cells

  • Bispecific antibodies simultaneously targeting ASPHD2 and immune effector cells

  • Intracellular antibody delivery systems to modulate ASPHD2 function directly

• Integrated multi-omics approaches: ASPHD2 antibodies will increasingly be incorporated into comprehensive disease profiling by:

  • Combining antibody-based proteomics with genomic and transcriptomic data

  • Integrating spatial information about ASPHD2 distribution with single-cell molecular profiles

  • Correlating ASPHD2 status with the broader epigenetic landscape in patient samples

• Machine learning integration: The data generated through ASPHD2 antibody applications will be leveraged through:

  • AI-assisted image analysis of ASPHD2 immunostaining patterns

  • Predictive modeling of patient outcomes based on ASPHD2 and other molecular features

  • Algorithm development for identifying optimal therapeutic strategies based on ASPHD2 status