HIPK2 Antibody, FITC conjugated

What is HIPK2 and why is it significant in research?

HIPK2 is a Y-regulated serine/threonine kinase originally identified for its capacity to interact with homeodomain transcription factors. It plays critical roles in multiple signaling pathways, including TP53, WNT/β-Catenin, TGF-β, Hippo, and Interferon pathways . As a pleiotropic modulator, HIPK2 influences seemingly contradictory biological events such as growth arrest and cell death, cell survival and proliferation, as well as morphogenesis and differentiation .

HIPK2's significance extends to its function as a tumor suppressor that controls cell proliferation by antagonizing LEF1/β-catenin-mediated transcription. Loss of HIPK2 leads to increased proliferative potential and expansion of the epidermal stem cell compartment, potentially contributing to tumorigenesis . These multifaceted roles make HIPK2 an important target for research across various biological contexts.

What are the main applications of FITC-conjugated HIPK2 antibodies?

FITC-conjugated HIPK2 antibodies are versatile tools with several key applications:

The FITC conjugation enables direct visualization without secondary antibodies, making these antibodies particularly valuable for multicolor staining protocols, live cell applications, and flow cytometry where direct detection reduces protocol complexity .

How do HIPK2 antibodies help in distinguishing between different HIPK2 isoforms?

HIPK2 exists in different splice variants, including full-length HIPK2 (HIPK2-FL) and a shorter isoform (HIPK2-S), which have distinct subcellular localizations and functions. While HIPK2-FL is involved in stress response, HIPK2-S localizes at the intercellular bridge where it phosphorylates histone H2B and spastin, both required for faithful cell division .

When selecting antibodies for isoform discrimination:

• Examine the epitope location - antibodies targeting regions unique to specific isoforms can differentiate between variants

• Consider Western blot validation - HIPK2-FL appears at approximately 131 kDa, while HIPK2-S presents at around 101 kDa

• Utilize immunofluorescence to observe distinct localization patterns - HIPK2-S specifically localizes to the intercellular bridge during cytokinesis

Researchers should carefully select antibodies based on the specific isoform they wish to study and validate detection using appropriate positive controls.

What are optimal conditions for using FITC-conjugated HIPK2 antibodies in flow cytometry?

For optimal results in flow cytometry applications with FITC-conjugated HIPK2 antibodies:

• Sample preparation: Use single-cell suspensions at concentrations of 1×10^6 cells/ml in PBS with 1% BSA and 0.1% sodium azide.

• Fixation and permeabilization: Since HIPK2 has both nuclear and cytoplasmic localization, use a fixation/permeabilization protocol suitable for intracellular antigens:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 or commercial permeabilization buffers

• Antibody dilution: Use the FITC-conjugated HIPK2 antibody at 1:20-1:100 dilution as recommended for flow cytometry applications

• Controls: Include an isotype control (FITC-conjugated rabbit IgG) at the same concentration to assess non-specific binding

• Compensation: When performing multicolor analysis, properly compensate for spectral overlap between FITC and other fluorophores

For quantifying stem cell populations where HIPK2 plays a regulatory role, consider using a protocol similar to that used in published research where cells were labeled with anti-α6-integrin antibody conjugated to FITC and anti-CD34 conjugated to Phycoerythrin .

How should researchers prepare samples for immunofluorescence studies using HIPK2-FITC antibodies?

For optimal immunofluorescence results with HIPK2-FITC antibodies:

• Cell preparation:

  • Grow cells on sterile coverslips or in chamber slides

  • At 60-80% confluence, wash cells with PBS

• Fixation options:

  • For preserving cytoskeletal elements: 4% paraformaldehyde for 15-20 minutes at room temperature

  • For nuclear proteins: methanol fixation (-20°C for 10 minutes)

  • When studying HIPK2's dual localization: test both methods to determine optimal detection

• Permeabilization:

  • Use 0.1-0.5% Triton X-100 in PBS for 5-10 minutes at room temperature

  • For gentler permeabilization: 0.1% saponin in PBS

• Blocking:

  • Block with 5% normal serum (from same species as secondary antibody if using indirect detection) with 0.3% Triton X-100 for 1 hour

• Antibody incubation:

  • Dilute FITC-conjugated HIPK2 antibody 1:50-1:200 in antibody dilution buffer

  • Incubate overnight at 4°C in a humidified chamber

  • Wash 3x with PBS

• Counterstaining:

  • Use DAPI (1μg/ml) for nuclear counterstaining

  • For co-staining with other proteins involved in HIPK2 pathways, select fluorophores with minimal spectral overlap with FITC

• Mounting:

  • Mount using an anti-fade mounting medium to prevent photobleaching of FITC

This protocol enables visualization of both nuclear and cytoplasmic HIPK2 localization, which is essential as HIPK2 shuttles between cellular compartments depending on its activation state.

What validation controls should be included when using HIPK2-FITC antibodies?

Rigorous validation is essential for ensuring reliable results with HIPK2-FITC antibodies:

• Positive controls:

  • Cell lines known to express HIPK2 (e.g., mouse kidney tissue, human renal cell carcinoma tissue)

  • Overexpression systems with HIPK2 expression vectors

• Negative controls:

  • HIPK2 knockout cell lines or tissues (if available)

  • HIPK2 knockdown cells using siRNA/shRNA

  • Primary antibody omission control

  • Isotype control (FITC-conjugated rabbit IgG) at equivalent concentration

• Specificity controls:

  • Peptide competition assay using the immunizing peptide (KLH-conjugated synthetic peptide derived from human HIPK2)

  • Detection of appropriately sized bands in Western blot (~131 kDa for HIPK2-FL and ~101 kDa for HIPK2-S)

• Cross-reactivity assessment:

  • Test antibody on various species samples to confirm expected reactivity with human and mouse samples

  • Validate reactivity against different HIPK family members to ensure specificity for HIPK2

• Functional validation:

  • Correlate antibody staining with functional assays (e.g., kinase activity assays)

  • Compare staining patterns with published literature on HIPK2 localization

These controls ensure that observed signals genuinely represent HIPK2 protein and not artifacts or cross-reactivity with other proteins.

How can FITC-conjugated HIPK2 antibodies be used to study HIPK2's role in transcriptional regulation?

HIPK2 acts as a transcriptional corepressor for several transcription factors, including SMAD1 and POU4F1/Brn3a . To investigate this regulatory function using HIPK2-FITC antibodies:

• Chromatin immunoprecipitation (ChIP) combined with immunofluorescence:

  • Perform ChIP using non-conjugated HIPK2 antibodies to pull down HIPK2-associated chromatin

  • Use FITC-conjugated HIPK2 antibodies to visualize HIPK2 recruitment to specific nuclear foci

  • This dual approach confirms the presence of HIPK2 at transcriptional regulatory sites

• Co-localization studies with transcription factors:

  • Use HIPK2-FITC antibodies alongside antibodies against known HIPK2-interacting transcription factors (e.g., LEF1/β-catenin)

  • Analyze co-localization in response to stimuli or during different cell cycle phases

  • Quantify the degree of co-localization using appropriate image analysis software

• Transcriptional activity correlation:

  • Combine HIPK2-FITC immunofluorescence with reporter gene assays

  • Correlate HIPK2 subcellular localization with repression of target genes like cyclin D1

  • This approach reveals how HIPK2 localization influences its transcriptional repressor function

• Dynamic studies of transcriptional complex formation:

  • Utilize FRAP (Fluorescence Recovery After Photobleaching) with HIPK2-FITC antibodies to study the dynamics of HIPK2 recruitment to transcriptional complexes

  • This technique provides insights into the kinetics of HIPK2's interaction with transcriptional machinery

These methodologies illuminate how HIPK2 functions as a corepressor of LEF1/β-catenin-mediated transcription, which is critical for its tumor suppressor activity .

What experimental approaches can be used to investigate HIPK2's interaction with β-catenin using fluorescent antibodies?

HIPK2 suppresses LEF1/β-catenin-mediated transcriptional activation, particularly of cyclin D1 expression . To study this interaction:

• Proximity ligation assay (PLA):

  • Use HIPK2-FITC antibody in combination with β-catenin antibody

  • PLA provides visualization of protein-protein interactions within 40nm distance

  • Quantify PLA signals to assess the extent of interaction under different conditions

• FRET (Förster Resonance Energy Transfer) analysis:

  • Use HIPK2-FITC antibody as donor and β-catenin labeled with an appropriate acceptor fluorophore

  • Measure energy transfer to confirm direct interaction and calculate proximity

  • This approach provides spatial resolution beyond conventional co-localization

• Co-immunoprecipitation followed by immunofluorescence:

  • Perform co-IP to confirm physical interaction between HIPK2 and β-catenin

  • Use immunofluorescence with HIPK2-FITC antibodies to visualize subcellular locations where interaction occurs

  • This combined approach links biochemical interaction with spatial information

• Live cell imaging of complex formation:

  • Use cell-permeable FITC-conjugated HIPK2 antibodies in live cells

  • Monitor recruitment of HIPK2 to β-catenin complexes in real-time

  • This approach reveals dynamic aspects of the interaction

• Deletion mutant analysis:

  • Express HIPK2 deletion mutants lacking the C-terminal YH domain, which is critical for recruiting the transcriptional corepressor CtBP

  • Use HIPK2-FITC antibodies to visualize how these mutations affect co-localization with β-catenin

  • This strategy helps map domains essential for the interaction

These approaches provide complementary information about the spatial, temporal, and functional aspects of HIPK2's interaction with β-catenin in suppressing transcriptional activation.

How can researchers use HIPK2 antibodies to examine its role in epidermal stem cell regulation?

HIPK2 controls the number of stem and progenitor cells in the skin, with loss of HIPK2 leading to expansion of the epidermal stem cell compartment . To investigate this role:

• Stem cell identification and quantification:

  • Use HIPK2-FITC antibodies in combination with epidermal stem cell markers (e.g., α6-integrin and CD34)

  • Flow cytometry analysis to quantify changes in stem cell populations in normal vs. HIPK2-deficient tissues

  • Calculate percentages of stem cells and correlate with HIPK2 expression levels

• Lineage tracing experiments:

  • Combine HIPK2 immunostaining with BrdU pulse-chase experiments

  • Track stem cell divisions and differentiation patterns in relation to HIPK2 expression

  • This approach reveals how HIPK2 influences stem cell fate decisions

• Colony-forming assays:

  • Sort cells based on HIPK2 expression using HIPK2-FITC antibodies and flow cytometry

  • Compare colony-forming efficiency and size between HIPK2-high and HIPK2-low populations

  • This functional assay connects HIPK2 expression to stem cell proliferative potential

• 3D organoid cultures:

  • Use immunofluorescence with HIPK2-FITC antibodies in 3D epidermal organoids

  • Analyze HIPK2 expression patterns across different cellular layers

  • Correlate with markers of proliferation and differentiation

• Cell cycle analysis in stem cell populations:

  • Combine HIPK2-FITC staining with cell cycle markers and stem cell markers

  • Perform multiparameter flow cytometry to assess how HIPK2 regulates cell cycle progression specifically in stem cells

  • This approach connects HIPK2's role in cell cycle regulation to its function in stem cell maintenance

These methodologies help elucidate how HIPK2 controls epidermal stem cell numbers by regulating proliferation through suppression of cyclin D1 expression .

What are common issues with FITC-conjugated HIPK2 antibodies and their solutions?

When working with FITC-conjugated HIPK2 antibodies, researchers may encounter several challenges:

When troubleshooting, always include appropriate controls and optimize each step of the protocol for your specific experimental system.

How should researchers interpret differential localization patterns of HIPK2?

HIPK2 shows dynamic subcellular localization that can vary based on cell type, cell cycle stage, and cellular stress conditions. When interpreting localization patterns:

• Nuclear vs. cytoplasmic localization:

  • Nuclear HIPK2 often indicates active transcriptional repression functions

  • Cytoplasmic HIPK2 may reflect roles in cytoplasmic signaling or could indicate regulation of HIPK2 activity

  • HIPK2 shuttles between nucleus and cytoplasm in response to cellular signals

• Subnuclear structures:

  • HIPK2 can concentrate in nuclear speckles or other subnuclear domains

  • Such patterns may indicate association with specific transcriptional complexes

  • Compare patterns with known nuclear domain markers to identify specific structures

• Isoform-specific patterns:

  • HIPK2-S specifically localizes at the intercellular bridge during cytokinesis

  • HIPK2-FL shows different localization patterns associated with stress response

  • Confirm which isoform you're detecting based on antibody epitope specificity

• Context-dependent interpretation:

  • In tumor samples: reduced nuclear HIPK2 may correlate with loss of tumor suppressor function

  • During development: changing localization patterns may reflect developmental regulation

  • During cell cycle: localization changes may indicate cell cycle-specific functions

• Colocalization analysis:

  • Quantify colocalization with interaction partners like LEF1/β-catenin

  • Calculate Pearson's or Mander's coefficients for objective assessment

  • Compare colocalization patterns between normal and pathological states

By carefully analyzing these patterns and correlating them with functional data, researchers can gain insights into HIPK2's diverse roles in different cellular contexts.

How can HIPK2-FITC antibodies be used in co-localization studies with WNT/β-catenin pathway proteins?

HIPK2 functions as a repressor of β-catenin-mediated transcription . To study these interactions:

• Triple co-localization analysis:

  • Use HIPK2-FITC antibody with antibodies against β-catenin and LEF1/TCF

  • Select compatible fluorophores (e.g., FITC for HIPK2, Cy3 for β-catenin, Cy5 for LEF1)

  • Analyze nuclear co-localization at transcriptionally active sites

• Pathway activation studies:

  • Treat cells with WNT pathway activators (e.g., LiCl, WNT3a)

  • Track changes in HIPK2 localization relative to β-catenin

  • Correlate with expression of WNT target genes like cyclin D1

• Super-resolution microscopy:

  • Use techniques like STORM or STED with HIPK2-FITC antibodies

  • Achieve nanometer-scale resolution of protein complexes

  • Determine precise spatial relationships between HIPK2 and WNT pathway components

• Time-course analysis after pathway modulation:

  • Activate or inhibit WNT signaling

  • Collect samples at multiple timepoints

  • Use HIPK2-FITC antibodies to track dynamic changes in localization and complex formation

• Transcriptional reporter correlation:

  • Combine immunofluorescence with TOPFlash WNT reporter assays

  • Correlate HIPK2 recruitment to β-catenin with suppression of reporter activity

  • This links localization patterns to functional outcomes

These approaches help elucidate how HIPK2 controls cell proliferation by repressing β-catenin-mediated transcription of target genes like cyclin D1, which is critical for its tumor suppressor function .

What experimental designs are recommended for studying HIPK2's role in tumor suppression?

HIPK2 functions as a tumor suppressor, with loss of HIPK2 leading to increased susceptibility to tumorigenesis . To investigate this role:

• Comparative expression analysis in normal vs. tumor tissues:

  • Use HIPK2-FITC antibodies for immunofluorescence on tissue microarrays

  • Quantify expression levels and correlate with tumor grade/stage

  • Analyze subcellular localization patterns as potential prognostic indicators

• Genetic modulation studies:

  • Create HIPK2 knockdown and overexpression models

  • Use HIPK2-FITC antibodies to confirm expression changes

  • Assess impacts on proliferation, cell cycle progression, and tumorigenic potential

• Cell cycle analysis:

  • Combine HIPK2-FITC staining with cell cycle markers

  • Perform flow cytometry to analyze how HIPK2 expression affects G1-S transition

  • This approach connects HIPK2 to cyclin D1 regulation and cell cycle control

• Signaling pathway integration:

  • Use HIPK2-FITC antibodies to study how HIPK2 interacts with multiple pathways (WNT/β-catenin, p53, TGF-β)

  • Analyze pathway crosstalk through multi-parameter immunofluorescence

  • This reveals how HIPK2 coordinates different tumor suppressor mechanisms

• In vivo tumor models:

  • Utilize mouse models with varied HIPK2 expression (wild-type, Hipk2+/-, Hipk2-/-)

  • Apply carcinogenesis protocols as described in the literature

  • Use HIPK2-FITC antibodies for tissue analysis to correlate expression with tumor progression

These comprehensive approaches provide insights into HIPK2's tumor suppressor mechanisms, particularly its role in controlling proliferation through antagonizing LEF1/β-catenin-mediated transcription of cyclin D1 .