HIPK3 Antibody

What is HIPK3 and why is it significant in research?

Homeodomain-interacting protein kinase 3 (HIPK3) is a conserved serine/threonine kinase that regulates various transcription factors influencing critical developmental processes, including cell proliferation, differentiation, apoptosis, and inflammatory responses. HIPK3 shares several functional domains with HIPK2, though its conservation is relatively low compared to other family members. Its significance in research stems from its diverse roles in cellular processes and implications in various pathological conditions, including cancer, neurodegeneration, and autoimmune disorders . Understanding HIPK3 function provides insights into fundamental cellular mechanisms and potential therapeutic targets.

What are the common specifications of commercial HIPK3 antibodies?

HIPK3 antibodies are typically available in various formats targeting different epitopes of the protein. Common specifications include:

• Host organisms: Predominantly rabbit-derived polyclonal antibodies

• Target specificity: Various regions including C-terminal (AA 1127-1156), internal regions (AA 841-1130), and other specific domains (AA 770-850, AA 401-500)

• Reactivity: Most commonly reactive with human samples, with many cross-reactive to mouse and rat

• Applications: Western blotting (WB), immunohistochemistry on paraffin-embedded sections (IHC-P), immunofluorescence (IF), ELISA, and immunocytochemistry (ICC)

• Conjugation options: Available as unconjugated antibodies or conjugated with HRP, FITC, or biotin for specific detection methods

What are the recommended applications for HIPK3 antibodies?

HIPK3 antibodies can be effectively utilized in multiple experimental applications depending on research objectives:

• Western blotting: For protein expression quantification and molecular weight confirmation

• Immunohistochemistry: For examining tissue localization patterns in paraffin-embedded or frozen sections

• Immunofluorescence: For subcellular localization studies in cultured cells

• ELISA: For quantitative detection of HIPK3 in solution

• RNA immunoprecipitation assays: For investigating HIPK3's interactions with RNA components, particularly important when studying circHIPK3 functions

The choice of application should be guided by the specific antibody's validated performance characteristics and the experimental question being addressed.

How should HIPK3 antibodies be validated for experimental use?

Proper validation of HIPK3 antibodies is critical for ensuring experimental reliability. A comprehensive validation approach should include:

• Specificity testing: Western blot analysis demonstrating a single band at the expected molecular weight (~130-140 kDa for full-length HIPK3)

• Positive and negative controls: Using tissues/cells known to express HIPK3 (positive) versus those with knockdown/knockout of HIPK3 (negative)

• Cross-reactivity assessment: Testing against closely related proteins (HIPK1, HIPK2, HIPK4) to confirm specificity

• Application-specific validation: For IHC applications, comparing staining patterns with mRNA expression data

• Lot-to-lot consistency: Evaluating performance consistency across different production lots

Researchers should also consider validating antibodies in their specific experimental system before conducting full-scale experiments to ensure optimal performance in the particular context of their research.

What are the optimal conditions for using HIPK3 antibodies in Western blotting?

For optimal Western blotting results when using HIPK3 antibodies, researchers should consider the following protocol parameters:

• Sample preparation: Total protein extraction with RIPA buffer containing protease inhibitors

• Protein loading: 20-50 μg of total protein per lane

• Gel percentage: 8-10% SDS-PAGE for optimal separation of the high molecular weight HIPK3 protein

• Transfer conditions: Wet transfer at 100V for 90-120 minutes or overnight at 30V for efficient transfer of large proteins

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody dilution: Typically 1:500-1:2000 (antibody-dependent, should be empirically determined)

• Incubation conditions: Overnight at 4°C with gentle rocking

• Washing: 3-5 washes with TBST, 5-10 minutes each

• Detection system: HRP-conjugated secondary antibodies with enhanced chemiluminescence detection

Optimization of these conditions may be necessary depending on the specific antibody and sample type.

What are the key considerations for immunohistochemistry using HIPK3 antibodies?

When performing immunohistochemistry with HIPK3 antibodies, researchers should address these critical factors:

• Fixation method: 10% neutral buffered formalin is typically suitable, though some epitopes may require gentler fixatives

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Blocking endogenous peroxidase: 3% hydrogen peroxide for 10 minutes

• Blocking non-specific binding: 5-10% normal serum from the species of the secondary antibody

• Primary antibody dilution: Typically 1:100-1:500 (requires optimization)

• Incubation time: Overnight at 4°C or 1-2 hours at room temperature

• Detection system: Compatible with standard immunohistochemistry detection systems (ABC, polymer-based)

• Counterstaining: Hematoxylin for nuclear visualization

• Controls: Include positive, negative, and isotype controls to validate staining specificity

The staining pattern should be interpreted in the context of known HIPK3 expression patterns and localization (predominantly nuclear with some cytoplasmic distribution).

How is HIPK3 implicated in rheumatoid arthritis, and how can antibodies help study this connection?

HIPK3 demonstrates significant relevance to rheumatoid arthritis (RA) research through its epigenetic regulation and relationship with inflammatory markers:

• Methylation status: HIPK3 shows hypomethylation in the blood of RA patients compared to osteoarthritis patients and healthy controls (p= 1.143×10^-8, FDR= 2.799×10^-7)

• Biomarker potential: HIPK3 methylation status shows high predictive power for RA diagnosis (AUC=0.829), especially valuable when combined with other clinical markers (AUC=0.864 when combined with ACPA- and RF+)

• Inflammation correlation: HIPK3 methylation levels are negatively correlated with C-reactive protein (CRP; r= -0.16, p= 0.01), a key inflammatory marker in RA

HIPK3 antibodies can be utilized to investigate protein expression patterns in synovial tissues and peripheral blood mononuclear cells from RA patients. This can be correlated with methylation data to establish a comprehensive understanding of HIPK3's role in RA pathogenesis. Antibody-based immunoprecipitation followed by mass spectrometry can also help identify HIPK3 interaction partners in inflammatory conditions .

What role does HIPK3 play in cancer research, and how can antibodies facilitate these studies?

HIPK3 demonstrates complex and context-dependent roles in cancer biology:

• Expression patterns: HIPK3 shows significantly downregulated mRNA and protein expression in non-small cell lung cancer (NSCLC) tissues

• Functional impact: HIPK3 silencing promotes invasion and metastasis in NSCLC models

• Prognostic value: Low HIPK3 expression correlates with poor survival in NSCLC patients

• Contrasting roles: While acting as a tumor suppressor in lung cancer, HIPK3 (particularly circHIPK3) may promote growth and metastasis in esophageal squamous cell carcinoma

HIPK3 antibodies enable several experimental approaches in cancer research:

• Tissue microarray analysis: Evaluating HIPK3 expression across tumor stages and grades

• Correlation studies: Examining relationships between HIPK3 protein levels and patient outcomes

• Functional studies: Using antibodies to validate HIPK3 knockdown or overexpression in cell models

• Mechanistic investigations: Immunoprecipitation to identify cancer-specific interaction partners

These applications collectively help elucidate HIPK3's role in tumorigenesis and its potential as a biomarker or therapeutic target.

How can HIPK3 antibodies be used to study neurodegenerative disorders, particularly Huntington's disease?

HIPK3 has emerged as a significant factor in Huntington's disease (HD) pathogenesis, presenting opportunities for antibody-based investigations:

• Autophagy regulation: HIPK3 functions as a novel kinase regulator of autophagy in HD cells, contributing to protein accumulation and disease progression

• Therapeutic implications: Inhibition of HIPK3 or loss of its kinase activity lowers mutant HTT levels via autophagy

• Model systems: HIPK3's role has been investigated in HD mouse primary neurons, human iPSC-derived neurons, and HD fly models

HIPK3 antibodies can facilitate HD research through:

• Protein localization studies: Determining HIPK3 distribution in affected neural tissues

• Co-localization analysis: Examining spatial relationships between HIPK3 and aggregated mutant HTT

• Quantification of expression changes: Monitoring HIPK3 levels during disease progression and in response to potential therapeutics

• Target engagement studies: Verifying the efficacy of HIPK3 inhibitors like AST487 in reducing HIPK3 activity

• Phosphoproteomic analysis: Identifying HIPK3 substrates in neuronal cells using phospho-specific antibodies

These applications provide critical insights into the mechanisms linking HIPK3 to HD pathogenesis and potential therapeutic strategies.

How can circular RNA forms of HIPK3 (circHIPK3) be distinguished from linear HIPK3 in experimental workflows?

Distinguishing circular RNA forms of HIPK3 (circHIPK3) from linear HIPK3 mRNA requires specialized approaches:

Experimental strategies:

• RNase R treatment: CircRNAs are resistant to RNase R digestion due to their lack of free ends, while linear RNAs are degraded

• Divergent primers: PCR with primers designed to amplify outward from a junction site will only amplify circular forms

• Northern blotting: Using junction-spanning probes to specifically detect circHIPK3

• RNA immunoprecipitation (RIP): Using antibodies against proteins that preferentially bind circRNAs

Analysis considerations:

• RNA-seq data analysis: Special bioinformatic pipelines (e.g., CIRCexplorer, CIRI) are required to detect back-spliced junction reads characteristic of circRNAs

• Junction verification: Sanger sequencing to confirm the exact back-splicing junction of circHIPK3

For functional studies, researchers should employ targeted knockdown strategies using siRNAs directed specifically at the back-splice junction of circHIPK3 to avoid affecting linear HIPK3 expression. This approach has been successfully used in studies examining circHIPK3's role in cardiac dysfunction and cancer models .

What are the best approaches for studying HIPK3 phosphorylation targets and kinase activity?

Investigating HIPK3's kinase function and substrate specificity requires sophisticated biochemical and cellular approaches:

In vitro kinase assays:

• Recombinant protein production: Express and purify active HIPK3 kinase domain using bacterial or mammalian expression systems

• Substrate screening: Utilize peptide arrays or protein microarrays to identify potential phosphorylation targets

• Validation assays: Perform in vitro kinase reactions with $γ-^32^P$ATP or non-radioactive ATP analogs

• Phosphosite mapping: Use mass spectrometry to identify specific residues phosphorylated by HIPK3

Cellular approaches:

• Phospho-specific antibodies: Develop antibodies against known or predicted HIPK3 phosphorylation sites

• Phosphoproteomic analysis: Compare phosphopeptide profiles between wild-type and HIPK3-deficient cells

• Kinase-dead mutants: Express catalytically inactive HIPK3 (typically K226M mutation) as a dominant-negative control

• Chemical genetics: Engineer analog-sensitive HIPK3 mutants that can utilize modified ATP analogs for specific labeling of substrates

Inhibitor studies:

• Small molecule inhibitors: Utilize HIPK3 inhibitors like AST487 to block kinase activity

• Dose-response relationships: Establish concentration-dependent effects on substrate phosphorylation

• Selectivity profiling: Determine inhibitor specificity across related kinases

These approaches collectively provide a comprehensive understanding of HIPK3's enzymatic function and biological targets.

How should researchers address contradictory findings in the literature regarding HIPK3 function in different disease models?

The divergent roles reported for HIPK3 across different disease models present a significant challenge for researchers. A systematic approach to reconciling contradictory findings should include:

Methodological standardization:

• Antibody validation: Ensure consistent use of validated HIPK3 antibodies with confirmed specificity

• Expression analysis: Employ multiple methods (qPCR, Western blot, immunohistochemistry) to verify HIPK3 expression patterns

• Isoform specificity: Distinguish between different HIPK3 isoforms and circular RNA forms in analyses

Context-dependent considerations:

• Tissue specificity: Acknowledge that HIPK3 may have different functions in different tissues (e.g., tumor suppressor in lung vs. oncogenic in esophageal cancer)

• Disease stage: Consider temporal aspects of disease progression when comparing studies

• Experimental systems: Distinguish between findings from cell lines, primary cultures, animal models, and human samples

Integrative approaches:

• Meta-analysis: Systematically review published literature with attention to methodological details

• Multi-omics integration: Combine transcriptomic, proteomic, and functional data to build comprehensive models

• Collaboration: Establish research consortia to standardize protocols and directly compare results across laboratories

Mechanistic resolution:

• Pathway analysis: Map HIPK3 functions to specific signaling pathways in each context

• Interactome studies: Identify context-specific protein interaction partners

• Genetic background: Consider the influence of genetic modifiers on HIPK3 function

By addressing these factors systematically, researchers can develop more nuanced models of HIPK3 function that account for apparently contradictory observations.

How can HIPK3 antibodies be integrated into multiplexed imaging approaches for complex tissue analysis?

Integrating HIPK3 antibodies into multiplexed imaging workflows enables simultaneous visualization of multiple markers, providing richer contextual information:

Advanced multiplexing strategies:

• Sequential immunofluorescence: Iterative staining, imaging, and antibody stripping/quenching

• Spectral unmixing: Using spectrally distinct fluorophores with computational separation of overlapping signals

• Mass cytometry imaging (IMC): Metal-tagged antibodies detected by laser ablation and mass spectrometry

• DNA-barcoded antibodies: Antibodies conjugated to DNA oligonucleotides for sequential detection

HIPK3 antibody considerations for multiplexing:

• Clone selection: Choose HIPK3 antibody clones compatible with fixation methods required for multiplexing

• Signal amplification: Consider tyramide signal amplification for low-abundance HIPK3 detection

• Antibody validation: Verify that multiplexing conditions do not alter HIPK3 epitope accessibility

• Cross-reactivity testing: Ensure no cross-reactivity between HIPK3 antibodies and other antibodies in the panel

Data analysis approaches:

• Spatial statistics: Quantify co-localization between HIPK3 and other markers

• Cell type identification: Combine HIPK3 with cell type-specific markers to identify expression patterns

• Neighborhood analysis: Examine spatial relationships between HIPK3-expressing cells and their microenvironment

These approaches are particularly valuable for investigating HIPK3's role in complex tissues such as inflammatory synovium in rheumatoid arthritis or heterogeneous tumor microenvironments .

What strategies can researchers employ to develop isoform-specific antibodies for HIPK3?

Developing antibodies that specifically recognize different HIPK3 isoforms requires targeted approaches:

Epitope selection strategies:

• Junction-spanning epitopes: Design peptide immunogens that span unique exon-exon junctions specific to particular isoforms

• Isoform-unique regions: Target sequences present exclusively in certain isoforms

• Post-translational modifications: Generate antibodies against modifications specific to certain isoforms

Production and screening workflow:

• Immunization: Use synthetic peptides or recombinant protein fragments as immunogens

• Hybridoma screening: Employ differential screening against multiple isoforms to identify clone specificity

• Recombinant antibody technology: Use phage display libraries with isoform-specific selection strategies

• Validation: Test against cells overexpressing individual isoforms and in isoform-specific knockdown models

Application-specific considerations:

• Western blotting: Optimize gel percentage to resolve isoforms with small molecular weight differences

• Immunoprecipitation: Verify pull-down of specific isoforms by mass spectrometry

• Immunohistochemistry: Validate specificity using tissues with known isoform expression patterns

Developing such isoform-specific antibodies would enable more precise characterization of HIPK3 biology, particularly in contexts where different isoforms may have distinct or even opposing functions .

How can single-cell approaches incorporate HIPK3 antibodies to understand cellular heterogeneity in disease models?

Single-cell technologies provide powerful tools for understanding HIPK3 expression and function at unprecedented resolution:

Single-cell protein analysis methods:

• Mass cytometry (CyTOF): Metal-tagged HIPK3 antibodies enable quantification across millions of individual cells

• Single-cell Western blotting: Microfluidic platforms for protein separation and antibody detection in individual cells

• Imaging mass cytometry: Combines mass cytometry with tissue imaging for spatial single-cell analysis

• Proximity extension assays: Antibody pairs with attached oligonucleotides for highly sensitive protein detection

Integration with transcriptomics:

• CITE-seq: Antibody-oligonucleotide conjugates enable simultaneous measurement of protein and mRNA

• Single-cell RNA-seq with protein validation: Follow-up validation of transcriptomic clusters using HIPK3 antibodies

• Spatial transcriptomics with protein co-detection: Combining in situ RNA analysis with antibody staining

Analysis and interpretation:

• Trajectory analysis: Map HIPK3 expression changes during cellular differentiation or disease progression

• Heterogeneity quantification: Identify subpopulations with distinct HIPK3 expression or activation states

• Regulatory network inference: Correlate HIPK3 with other proteins to infer functional relationships

These approaches are particularly valuable for understanding the complex roles of HIPK3 in heterogeneous disease contexts, such as the inflammatory microenvironment in rheumatoid arthritis or the diverse cell populations affected in neurodegenerative conditions .