HIPK2 Antibody, Biotin conjugated

What is HIPK2 and what cellular functions does it regulate?

HIPK2 (Homeodomain-interacting protein kinase 2) is a serine/threonine kinase that plays critical roles in transcriptional regulation, growth control, and apoptotic pathways. The protein is activated in response to DNA damage, including UV radiation and chemotherapeutic drugs. Upon activation, HIPK2 phosphorylates p53 at Ser46, promoting the transcription of pro-apoptotic p53 target genes . Additionally, HIPK2 interacts with numerous transcription factors that control developmental processes, tumor suppression, and apoptosis mechanisms . Structurally, HIPK2 has a calculated molecular weight of 131 kDa, though it can be observed at both 131 kDa and 101 kDa in experimental systems .

What are the primary research applications for HIPK2 antibodies?

HIPK2 antibodies have been validated for multiple experimental applications including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunoprecipitation (IP), Co-Immunoprecipitation (CoIP), and Enzyme-Linked Immunosorbent Assay (ELISA) . Published literature demonstrates successful application in knockdown/knockout studies, with at least 8 publications reporting WB applications, 3 for IHC, and others for IF, IP, and CoIP techniques . For biotin-conjugated HIPK2 antibodies specifically, ELISA stands as the primary validated application, though researchers may develop protocols for other detection systems leveraging the biotin-streptavidin interaction .

What is the advantage of using a biotin-conjugated HIPK2 antibody?

Biotin-conjugated HIPK2 antibodies offer several methodological advantages in research applications. The biotin-streptavidin system provides one of the strongest non-covalent biological interactions known, with high affinity binding (Kd ≈ 10^-15 M). This property enables sensitive detection methods as the biotin tag can be recognized by streptavidin conjugated to various reporter molecules such as enzymes (HRP, AP) or fluorophores . In ELISA applications specifically, biotin-conjugated HIPK2 antibodies work within a sandwich format where they bridge between plate-bound primary antibodies and enzyme-conjugated avidin/streptavidin systems, allowing for highly sensitive quantification of HIPK2 in complex biological samples .

How should biotin-conjugated HIPK2 antibodies be stored to maintain optimal activity?

Biotin-conjugated HIPK2 antibodies require specific storage conditions to maintain their structural integrity and immunoreactivity. According to manufacturer specifications, these antibodies should be stored at -20°C or -80°C immediately upon receipt . The storage buffer typically contains preservatives such as 0.03% Proclin 300 and stabilizers including 50% glycerol in 0.01M PBS at pH 7.4 . Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of immunoreactivity . For working solutions, aliquoting the antibody into single-use volumes prior to freezing is recommended. When handling the antibody, researchers should maintain sterile conditions and avoid contamination that could compromise experimental results.

What are the recommended dilution ratios for different experimental applications?

Dilution ratios for HIPK2 antibodies vary significantly based on the specific application and antibody formulation. For non-conjugated HIPK2 antibodies, Western Blot applications typically require dilutions ranging from 1:200 to 1:1000, while immunohistochemistry protocols recommend dilutions between 1:500 and 1:2000 . For biotin-conjugated HIPK2 antibodies in ELISA applications, optimal dilutions must be determined empirically for each specific experimental system . Researchers should perform a titration series during assay development to identify the dilution that provides the optimal signal-to-noise ratio. Importantly, these recommendations serve as starting points, and assay-specific optimization is essential since signal strength depends on multiple factors including target abundance, sample type, and detection system sensitivity .

How can cross-reactivity be assessed when using HIPK2 antibodies across different species?

Assessment of cross-reactivity is critical when designing experiments involving multiple species. According to product information, commercially available HIPK2 antibodies demonstrate varying species reactivity profiles. Some antibodies, such as product 55408-1-AP, show reactivity with human, mouse, and rat samples , while others like CSB-PA867138LD01HU are specifically reactive with human HIPK2 .

To validate cross-reactivity:

• Begin with literature review and manufacturer data to identify expected cross-reactivity.

• Perform preliminary Western blot analysis using positive control samples from each species of interest.

• Include appropriate negative controls such as HIPK2-knockout tissue or HIPK2-depleted cell lysates.

• Verify band specificity by comparing observed molecular weights to predicted values (131 kDa for full-length HIPK2).

• For definitive validation, consider epitope mapping or mass spectrometry analysis of immunoprecipitated proteins.

Cross-reactivity validation is particularly important for studies comparing HIPK2 function across evolutionary models or when translating findings between animal models and human samples .

How can researchers optimize ELISA protocols using biotin-conjugated HIPK2 antibodies?

Optimization of ELISA protocols using biotin-conjugated HIPK2 antibodies requires systematic assessment of multiple parameters. The standard sandwich ELISA format employs a capture antibody pre-coated on the microplate, followed by sample addition, biotin-conjugated detection antibody, and enzyme-conjugated avidin .

Key optimization steps include:

• Antibody pair selection: Ensure the capture and detection antibodies recognize non-overlapping epitopes on HIPK2.

• Blocking optimization: Test different blocking agents (BSA, casein, non-fat milk) at various concentrations to minimize background signal.

• Sample preparation: Optimize lysis buffers and determine whether pre-clearing steps are needed for complex samples.

• Incubation conditions: Systematically test temperature (4°C, room temperature, 37°C) and duration variables.

• Signal development: Optimize substrate concentration and development time to achieve maximum sensitivity without signal saturation.

The TMB substrate system is commonly used with HRP-conjugated avidin, with absorbance measured at 450 nm . Standard curves should be prepared using recombinant HIPK2 protein at concentrations spanning the expected sample range, typically using 7-8 points with 2-fold dilutions .

What controls are essential when using biotin-conjugated HIPK2 antibodies in research applications?

Implementing comprehensive controls is critical for ensuring experimental validity when working with biotin-conjugated HIPK2 antibodies:

• Positive controls: Include samples with known HIPK2 expression such as mouse kidney tissue or specific cell lines with verified HIPK2 expression .

• Negative controls:

  • Isotype controls using non-specific rabbit IgG, biotin-conjugated

  • HIPK2 knockout or knockdown samples when available

  • Secondary reagent-only controls (omitting primary antibody)

• Specificity controls:

  • Pre-adsorption with recombinant HIPK2 protein

  • Comparison with alternative HIPK2 antibodies recognizing different epitopes

• Technical controls:

  • For ELISA: Standard curve prepared with recombinant HIPK2 protein (816-977AA region is commonly used for immunization)

  • For biotinylated systems: Endogenous biotin blocking when working with tissues known to contain high endogenous biotin (liver, kidney)

• Normalization controls: Include housekeeping proteins or total protein measurements to account for loading variations in quantitative applications .

Each control should be systematically evaluated to ensure signal specificity and experimental reproducibility.

How does HIPK2 phosphorylation status affect antibody recognition in different experimental contexts?

HIPK2 undergoes complex post-translational modifications that can significantly influence antibody epitope accessibility and recognition. Phosphorylation is particularly important as HIPK2 is activated through autophosphorylation and can be phosphorylated by upstream kinases in response to DNA damage signaling pathways .

Key considerations include:

• Epitope masking: Phosphorylation events, particularly within the activation loop, can alter protein conformation and potentially mask antibody binding sites.

• Phospho-specific detection: When studying HIPK2 activation, researchers should consider whether their antibody recognizes total HIPK2 or specific phosphorylated forms.

• Sample preparation impact: Phosphatase inhibitors must be included in lysis buffers when studying phosphorylated HIPK2. Their omission may lead to rapid dephosphorylation and altered antibody recognition.

• Cellular context: UV radiation and chemotherapeutic drugs induce HIPK2 phosphorylation, potentially altering antibody reactivity between treated and untreated samples .

• Western blot considerations: Phosphorylated forms of HIPK2 may exhibit slightly altered migration patterns on SDS-PAGE gels, with the phosphorylated form typically migrating at a slightly higher apparent molecular weight than predicted.

Researchers studying HIPK2 activity rather than just expression levels should consider using activity assays in parallel with immunodetection methods for complete characterization.

What are common sources of background in assays using biotin-conjugated HIPK2 antibodies?

High background is a frequent challenge when working with biotin-conjugated antibodies, including those targeting HIPK2. Several mechanisms contribute to this issue:

• Endogenous biotin interference: Many mammalian tissues (particularly liver, kidney, and brain) contain high levels of endogenous biotin that can directly interact with detection reagents. This is particularly relevant when analyzing samples from these tissues .

• Non-specific binding: Insufficient blocking or suboptimal buffer compositions can lead to direct binding of biotin-conjugated antibodies to the solid phase.

• Cross-reactivity: Though antibodies may be affinity-purified (>95% purity using Protein G), residual cross-reactivity with structurally similar proteins can occur .

• Avidin/streptavidin binding to non-biotinylated components: Some negatively charged proteins can non-specifically interact with the positively charged avidin/streptavidin molecules.

• Improper washing: Inadequate washing between steps, particularly after the biotin-conjugated antibody incubation, can lead to residual signal development.

Mitigation strategies include:

• Pre-blocking endogenous biotin using unconjugated streptavidin or avidin

• Optimizing blocking agents (5% BSA is often effective)

• Using specialized blocking reagents designed for biotin-streptavidin systems

• Increasing wash stringency with detergent-containing buffers

• When analyzing problematic tissues, consider alternative detection systems

How can the specificity of HIPK2 detection be validated in experimental systems?

Validating HIPK2 antibody specificity is essential for generating reliable research data. A comprehensive validation approach includes:

• Genetic validation approaches:

  • HIPK2 knockdown using siRNA or shRNA

  • CRISPR/Cas9-mediated HIPK2 knockout cell lines

  • Comparison with published HIPK2 knockout mouse models

• Biochemical validation:

  • Western blot analysis confirming the correct molecular weight (131 kDa and 101 kDa bands)

  • Immunoprecipitation followed by mass spectrometry

  • Peptide blocking experiments using the immunizing peptide (e.g., amino acids 816-977 for some antibodies)

• Expression pattern correlation:

  • Comparison of results with published tissue expression patterns

  • Multi-antibody comparison using antibodies targeting different HIPK2 epitopes

  • Correlation of protein detection with mRNA expression data

• Functional validation:

  • Induction of HIPK2 expression/activity using DNA-damaging agents such as UV radiation

  • Detection of increased p53 Ser46 phosphorylation (a known HIPK2 substrate) correlating with HIPK2 activity

Evidence for antibody specificity has been documented in multiple publications, with at least 8 publications specifically validating HIPK2 antibodies in Western blot applications and 3 in immunohistochemistry applications .

What considerations should be made when conjugating HIPK2 antibodies to biotin in-house?

Researchers may choose to perform in-house biotinylation of HIPK2 antibodies using commercial conjugation kits. This approach requires careful consideration of multiple factors:

• Antibody preparation requirements:

  • The antibody must be in an amine-free buffer (e.g., HEPES, MES, MOPS, or phosphate) at pH 6.5-8.5

  • Avoid buffers containing nucleophilic components, thiols, glycine, or preservatives like Thiomersal

  • Optimal antibody concentration is 1-2.5 mg/ml in a volume of 40-100 μl (for 100-200 μg antibody)

• Buffer compatibility:

  • Common preservatives like sodium azide (0.02-0.1%) and EDTA do not interfere with conjugation

  • If the antibody contains incompatible components, buffer exchange is required prior to conjugation

• Conjugation chemistry optimization:

  • The molar ratio of biotin to antibody significantly impacts assay performance

  • Excessive biotinylation can disrupt antigen binding by modifying lysine residues within the antigen-binding site

  • Insufficient biotinylation reduces detection sensitivity

• Quality control after conjugation:

  • Perform ELISA comparing pre- and post-conjugation antibody activity

  • Assess degree of biotinylation using HABA assay or mass spectrometry

  • Verify retention of specificity through Western blot analysis

• Storage of conjugated antibody:

  • Store at -20°C in buffer containing 50% glycerol to prevent freeze-thaw damage

  • Add protein stabilizers (e.g., 0.1% BSA) for long-term stability

Commercial conjugation kits like the LYNX Rapid Plus Biotin (Type 2) Antibody Conjugation Kit provide optimized reagents and protocols for achieving high conjugation efficiency with 100% antibody recovery and no requirement for desalting or dialysis .

How can biotin-conjugated HIPK2 antibodies be used to study HIPK2's role in apoptotic pathways?

Biotin-conjugated HIPK2 antibodies provide valuable tools for investigating HIPK2's critical role in apoptotic signaling cascades. Experimental approaches include:

• ELISA-based quantification:

  • Measure HIPK2 protein levels in response to apoptotic stimuli

  • Compare HIPK2 expression between normal and apoptotic cells

  • Develop time-course experiments tracking HIPK2 induction following DNA damage

• Biotin-streptavidin based immunoprecipitation:

  • Capture HIPK2 protein complexes to identify interaction partners in apoptotic pathways

  • Investigate dynamic changes in protein interactions following apoptotic stimuli

  • Couple with mass spectrometry for unbiased interaction profiling

• Multiplexed detection systems:

  • Combine biotin-conjugated HIPK2 antibodies with fluorescently-labeled antibodies against apoptotic markers

  • Analyze co-localization of HIPK2 with p53 and other pro-apoptotic factors

  • Develop flow cytometry protocols to correlate HIPK2 expression with apoptotic cell populations

• Functional studies:

  • Correlate HIPK2 protein levels with its kinase activity through p53 Ser46 phosphorylation

  • Examine HIPK2 accumulation following inhibition of its degradation pathways

  • Investigate caspase-dependent cleavage of HIPK2 and the resulting enhancement of activity

When interpreting results, researchers should consider that HIPK2's regulation involves complex post-translational modifications including sumoylation and ubiquitination, which are affected by DNA damaging agents .

What are appropriate normalization strategies when quantifying HIPK2 in different tissue samples?

Accurate quantification of HIPK2 across tissue samples requires robust normalization strategies to account for biological and technical variability:

• Western blot normalization:

  • Housekeeping proteins (β-actin, GAPDH, tubulin) serve as loading controls

  • Total protein normalization using stain-free technology or Ponceau staining

  • For tissues with variable housekeeping protein expression, multiple reference proteins should be used

• ELISA normalization:

  • Express HIPK2 concentration relative to total protein concentration

  • Use purified recombinant HIPK2 to generate standard curves

  • Include identical control samples across multiple plates to account for inter-assay variability

• Tissue-specific considerations:

  • HIPK2 expression varies significantly across tissues, with particularly notable expression in kidney tissue

  • Baseline expression should be established for each tissue type

  • For IHC applications, positive control tissues include mouse kidney, brain, and heart tissue

• Experimental design factors:

  • Include biological replicates (n≥3) to account for natural variation

  • Technical replicates (typically duplicates or triplicates) should be averaged

  • Consider paired sample design when comparing treated vs. untreated conditions

• Statistical approaches:

  • Apply appropriate statistical tests based on data distribution

  • For non-normally distributed data, consider non-parametric tests

  • Calculate and report coefficients of variation to demonstrate assay reproducibility

These normalization strategies enable reliable comparison of HIPK2 expression across different experimental conditions, tissue types, and disease states.

How does HIPK2 detection in cancer research help understand tumor suppression mechanisms?

Biotin-conjugated HIPK2 antibodies provide valuable tools for cancer researchers investigating HIPK2's tumor suppressive functions:

• Clinical correlation studies:

  • HIPK2 expression analysis in tumor vs. adjacent normal tissue

  • Correlation of HIPK2 levels with tumor grade, stage, and patient outcomes

  • IHC applications have been validated in human gliomas and renal cell carcinoma tissues

• Mechanistic investigations:

  • HIPK2's phosphorylation of p53 at Ser46 promotes apoptosis in cancer cells

  • Detection of this phosphorylation event serves as a functional readout of HIPK2 activity

  • Loss of HIPK2 function is associated with resistance to chemotherapy and radiation

• Regulation of HIPK2 in cancer:

  • HIPK2 is regulated by both sumoylation and ubiquitination

  • DNA damaging agents inhibit ubiquitination and subsequent degradation of HIPK2

  • Caspase-dependent cleavage removes HIPK2's inhibitory domain, enhancing activity

• Therapeutic implications:

  • Restoration of HIPK2 function represents a potential therapeutic strategy

  • Monitoring HIPK2 levels may predict responsiveness to DNA-damaging therapies

  • HIPK2 status could serve as a biomarker for treatment selection

• Technical considerations for cancer sample analysis:

  • Tumor heterogeneity necessitates careful sampling strategies

  • Comparison of HIPK2 protein levels with mRNA expression data

  • Analysis of HIPK2 localization (nuclear vs. cytoplasmic) provides functional insights

These approaches utilizing biotin-conjugated HIPK2 antibodies have contributed significantly to understanding HIPK2's role in maintaining genomic integrity and preventing malignant transformation through regulation of critical tumor suppressor pathways .