HIPK2 Antibody, HRP conjugated

What is HIPK2 and what molecular weight should I expect when detecting it with an HRP-conjugated antibody?

HIPK2 is a serine/threonine kinase that functions as a corepressor inhibiting MEF2-dependent gene expression in undifferentiated myoblasts. It is constitutively associated with a multi-protein complex containing histone deacetylases HDAC3 and HDAC4 that serves to silence MEF2C-dependent transcription .

When detecting HIPK2 with an HRP-conjugated antibody via Western blotting, you should expect to observe:

• Full-length HIPK2: 130-140 kDa band

• Cleaved HIPK2: Approximately 101 kDa band

The observation of both bands is common during muscle differentiation or apoptosis, as HIPK2 undergoes caspase-mediated cleavage following aspartic acids 916 and 977, generating a C-terminally truncated protein with altered function .

What are the optimal sample preparation techniques for HIPK2 detection using HRP-conjugated antibodies?

For effective HIPK2 detection:

• Cell/Tissue Lysis Protocol:

  • Use lysis buffer containing phosphatase inhibitors if studying phosphorylated forms of HIPK2

  • Include protease inhibitors to prevent degradation of full-length HIPK2

  • For detecting HIPK2 interactions with MEF2C or HDACs, use gentle non-ionic detergents (0.5-1% NP-40 or Triton X-100)

• Sample Types with Validated Detection:

  • Mouse kidney tissue (confirmed positive for Western blotting)

  • Human, mouse, and rat samples have demonstrated reactivity

  • Cell lines with confirmed expression: C2C12 myoblasts

• Antibody Dilution Range:

  • Western Blotting: 1:200-1:1000 dilution is typically recommended

  • Titration may be necessary for optimal signal-to-noise ratio with HRP-conjugated versions

How do I distinguish between full-length HIPK2 and its cleaved form in experimental assays?

Distinguishing between full-length and cleaved HIPK2 is critical for studies of muscle differentiation and apoptosis:

• Western Blot Analysis:

  • Full-length HIPK2 appears at 130-140 kDa

  • Cleaved form appears at approximately 101 kDa after caspase-mediated cleavage at aspartic acids 916 and 977

  • Use antibodies that target different regions:

    • N-terminal antibodies (e.g., N6A10) will detect both forms

    • C-terminal antibodies (e.g., C1B3) will detect only full-length protein

• Functional Differences:

  • Full-length HIPK2 binds MEF2C, HDAC3, and HDAC4 in a repressive complex

  • Cleaved HIPK2 (1-916) has strongly impaired ability to bind MEF2C and HDAC3/4

  • Full-length HIPK2 can phosphorylate MEF2C efficiently

  • Cleaved HIPK2 shows impaired MEF2C phosphorylation capability

• Experimental Controls:

  • Include λ phosphatase treatment to confirm phosphorylation status differences

  • Use known inducers of HIPK2 cleavage (differentiation conditions or apoptotic stimuli) as positive controls

How can I effectively use HRP-conjugated HIPK2 antibodies to investigate protein-protein interactions in the MEF2-HDAC complex?

HIPK2 forms a multi-protein complex with MEF2C, HDAC3, and HDAC4 during transcriptional repression. To study these interactions:

• Co-Immunoprecipitation Protocol:

  • Primary immunoprecipitation with HIPK2 antibody followed by detection of associated proteins

  • Reciprocal IP with MEF2C or HDAC antibodies followed by HIPK2 detection

  • Data from C2C12 cells demonstrate that endogenous HIPK2 co-immunoprecipitates with MEF2, HDAC3, and HDAC4

• Sequential Detection Strategy:

  • Strip and reprobe membranes to detect multiple proteins from same IP sample

  • For HRP-conjugated antibodies, thorough stripping is essential to prevent residual signal

• Visualization of Complex Formation:

  • HIPK2 and its binding partners show nuclear colocalization

  • HIPK2 and nuclear HDAC4 demonstrate almost complete colocalization in nuclear speckles

  • Only a fraction of HDAC3 binds to and colocalizes with HIPK2, suggesting substoichiometric amounts in the complex

• Quantitative Analysis:

  • Densitometric analysis can reveal relative binding affinities

  • Preferential binding to HDAC4 compared to HDAC3 has been observed

What methodologies can I employ to study HIPK2 phosphorylation activity on MEF2C using HRP-conjugated antibodies?

HIPK2 phosphorylates MEF2C, affecting its transcriptional activity. Here's how to investigate this:

• Phosphorylation Assay Design:

  • Co-express HIPK2 (wild-type or kinase-dead K221A mutant) with MEF2C

  • Detect phosphorylated MEF2C by band shift in Western blot (slower migration)

  • Confirm phosphorylation by λ phosphatase treatment, which converts the slower migrating form to a faster migrating band

• Controls for Specificity:

  • Include kinase-inactive HIPK2 K221A as negative control

  • Use shRNA-mediated knockdown of HIPK2 to validate phosphorylation dependency

  • Wild-type HIPK2 induces phosphorylation of MEF2C, while kinase-dead mutant does not

• Analysis of HDAC4-Associated Kinase Activity:

  • HDAC4 expression alone triggers MEF2C phosphorylation

  • Expression of dominant-negative HIPK2 K221A dose-dependently reduces MEF2C phosphorylation

  • shRNA-mediated knockdown of HIPK2 significantly impairs HDAC4-associated kinase activity

• Quantification Methods:

  • Measure ratio of phosphorylated to unphosphorylated MEF2C

  • Compare phosphorylation levels across experimental conditions using densitometry

What approaches can I use to study the dynamic regulation of HIPK2 during muscle differentiation or apoptotic responses?

HIPK2 undergoes caspase-mediated cleavage during muscle differentiation and in response to DNA damage. To study this:

• Tracking HIPK2 Cleavage Kinetics:

  • Monitor the appearance of the 101 kDa cleaved form using Western blotting

  • Compare with markers of differentiation (myogenin, MyHC) or apoptosis (cleaved caspases)

  • During muscle differentiation, caspase activity increases, resulting in HIPK2 cleavage after aspartic acids 916 and 977

• Functional Analysis of Cleaved HIPK2:

  • Compare wild-type HIPK2 with truncated HIPK2 1-916 variant

  • Assess MEF2C phosphorylation capability (impaired in truncated form)

  • Evaluate MEF2C acetylation status (wild-type HIPK2 completely reverses CBP-triggered acetylation, truncated form only mildly reduces it)

  • Test protein-protein interactions (truncated form has strongly impaired ability to bind MEF2C, HDAC3, and HDAC4)

• Manipulation of HIPK2 Activity:

  • Use caspase inhibitors to prevent cleavage

  • Create non-cleavable HIPK2 mutants (D916A/D977A) to assess functional impacts

  • Apply DNA-damaging agents to modify HIPK2 activity and stability

• Signal Integration Analysis:

  • HIPK2 is activated by DNA damage including UV radiation and chemotherapeutic drugs

  • Upon activation, it phosphorylates p53 at Ser46 to promote transcription of pro-apoptotic target genes

  • Track these events in parallel with HIPK2 status

What are the optimal conditions for multiplexed detection of HIPK2 and its post-translational modifications?

For comprehensive analysis of HIPK2 regulation:

• Detection of Multiple PTMs:

  • Phosphorylation: Use phospho-specific antibodies or Phos-tag gels

  • Sumoylation: HIPK2 is regulated by sumoylation

  • Ubiquitination: HIPK2 undergoes ubiquitination leading to degradation, which is inhibited by DNA damage

• Multiplexed Immunodetection Strategy:

  • Sequential probing with antibodies against different modifications

  • For HRP-conjugated antibodies, complete stripping between detections is critical

  • Consider using antibodies from different host species for simultaneous detection

• Subcellular Localization Analysis:

  • HIPK2 shows distinct nuclear speckle localization

  • Co-staining with compartment markers can reveal regulatory dynamics

  • HIPK2 colocalizes with MEF2, HDAC3, and HDAC4 in the nucleus

• Integration with Functional Readouts:

  • Correlate HIPK2 modifications with transcriptional reporter assays

  • Monitor target gene expression (e.g., muscle-specific genes repressed by HIPK2)

  • shRNA-mediated downregulation of HIPK2 results in elevated expression of muscle-specific genes

What are common challenges when using HRP-conjugated HIPK2 antibodies and how can they be addressed?

• Specificity Concerns:

  • Cross-reactivity with related kinases

  • Solution: Validate using HIPK2 knockout/knockdown controls

  • The observed 130-140 kDa band should disappear in knockdown samples

• Detection Sensitivity:

  • Endogenous HIPK2 levels may be low in some cell types

  • Solution: Use enhanced chemiluminescence substrates with longer exposure times

  • Consider sample enrichment through immunoprecipitation before detection

• Multiple Band Patterns:

  • Full-length (130-140 kDa) and cleaved form (101 kDa) may appear simultaneously

  • Additional bands may represent degradation products or other post-translational modifications

  • Solution: Include appropriate controls (phosphatase treatment, caspase inhibitors)

• Signal Optimization:

  • For Western blotting: Try different blocking solutions (BSA vs. milk protein)

  • Antibody dilution optimization in range of 1:200-1:1000

  • Substrate selection based on signal intensity requirements

How can I design experiments to distinguish between HIPK2 isoforms or detect specific functional states?

• Isoform-Specific Detection:

  • Use region-specific antibodies (N-terminal vs. C-terminal)

  • N-terminal antibodies (e.g., N6A10) detect both full-length and cleaved forms

  • C-terminal antibodies (e.g., C1B3) detect only full-length protein

• Functional State Analysis:

  • Active HIPK2: Associated with phosphorylated binding partners like MEF2C

  • Repressive complex: Co-detection with HDAC3, HDAC4, and MEF2

  • Cleaved form (1-916): Has impaired ability to interact with partner proteins

• Experimental Manipulation:

  • Expression of wild-type vs. kinase-dead (K221A) HIPK2

  • Expression of non-cleavable HIPK2 mutants (D916A/D977A)

  • Induction of DNA damage to activate HIPK2

• Comparative Analysis Across Models:

  • HIPK2 antibodies have demonstrated reactivity in human, mouse, and rat samples

  • Positive detection confirmed in various tissues including kidney, brain, heart

What are the most effective imaging and quantification methods for HRP-conjugated HIPK2 antibody signals?

• Optimal Imaging Parameters:

  • Chemiluminescence: Multiple exposures to capture dynamic range

  • Digital imaging: Use of cooled CCD cameras for higher sensitivity and dynamic range

  • Exposure time optimization: Short for strong signals, longer for weak signals

• Quantification Best Practices:

  • Background subtraction using adjacent areas or negative control lanes

  • Normalization to loading controls (β-actin, GAPDH)

  • Linear range determination using standard curves with recombinant protein

• Comparative Analysis:

  • Relative quantification across experimental conditions

  • Ratio analysis of full-length vs. cleaved HIPK2

  • Correlation with functional outcomes (e.g., MEF2C phosphorylation levels, target gene expression)

• Data Presentation:

  • Include representative images with molecular weight markers

  • Present quantitative data as mean ± standard deviation from multiple experiments

  • Statistical analysis of differences between experimental conditions

How can HRP-conjugated HIPK2 antibodies be utilized to investigate its role in cancer and neurological disorders?

• Cancer Research Applications:

  • HIPK2 phosphorylates p53 at Ser46 to promote pro-apoptotic gene transcription

  • Detection in tumor samples: Validated in human gliomas and renal cell carcinoma tissues

  • Analysis of HIPK2 levels before and after chemotherapeutic treatment

• Neurological Disease Models:

  • Detection validated in brain tissues from humans, mice, and rats

  • Analysis of HIPK2 expression and modification patterns in neurodegenerative conditions

  • Correlation with markers of cellular stress and DNA damage

• Experimental Design Considerations:

  • Use tissue microarrays for high-throughput screening across multiple samples

  • Combine with markers of apoptosis, DNA damage, and tissue-specific differentiation

  • Correlate HIPK2 status with clinical outcomes or disease progression

• Therapeutic Target Validation:

  • Monitor HIPK2 modifications in response to experimental therapeutics

  • DNA-damaging agents inhibit ubiquitination and degradation of HIPK2

  • Track changes in HIPK2-dependent transcriptional programs

What approaches can be employed to study HIPK2 involvement in developmental processes and differentiation?

• Developmental Stage Analysis:

  • HIPK2 interacts with transcription factors controlling developmental processes

  • Temporal profiling of HIPK2 levels during embryonic development

  • Spatial mapping of expression patterns in developing tissues

• Myogenic Differentiation Model:

  • HIPK2 acts as a corepressor inhibiting MEF2-dependent gene expression in myoblasts

  • Caspase-mediated cleavage of HIPK2 occurs during differentiation, alleviating repression

  • Experimental timeline:

    • Monitor HIPK2 status across differentiation stages

    • Correlate with muscle-specific gene expression

    • Track formation and dissolution of repressive complexes

• Genetic Manipulation Strategies:

  • HIPK2 knockdown using shRNAs leads to elevated expression of muscle-specific genes

  • Expression of non-cleavable HIPK2 mutants can block normal differentiation

  • Rescue experiments with wild-type vs. modified HIPK2 constructs

• Integrated Multi-Omics Approach:

  • Combine HIPK2 protein analysis with transcriptomics of target genes

  • Correlate with epigenetic modifications (histone acetylation status)

  • Map HIPK2-dependent regulatory networks during differentiation

How can multiple detection methods be integrated for comprehensive HIPK2 functional analysis?

• Complementary Technique Integration:

  • Western blotting: Protein levels and post-translational modifications

  • Immunohistochemistry: Tissue localization and expression patterns

  • Co-immunoprecipitation: Protein-protein interactions

  • ChIP assays: Genomic binding sites of HIPK2-containing complexes

• Multi-modal Approach Benefits:

  • Cross-validation of findings across techniques

  • Comprehensive view of HIPK2 biology from molecular to cellular levels

  • Integration of protein status with functional outcomes

• Experimental Design Framework:

  Technique	Application	Recommended Antibody Dilution	Key Controls
  Western Blot	Protein levels, PTMs	1:200-1:1000	HIPK2 knockdown, phosphatase treatment
  IHC	Tissue expression	1:500-1:2000	Peptide competition, negative tissues
  Co-IP	Protein complexes	Application-specific	IgG control, input lysate
  IF	Subcellular localization	Validated in literature	Secondary-only control

• Data Integration Strategy:

  • Correlate HIPK2 levels/modifications with target gene expression

  • Link protein interaction data with functional outcomes

  • Develop predictive models of HIPK2-dependent regulatory networks