KCNK15 Antibody

What is KCNK15 and why is it significant in research?

KCNK15 (also known as TASK-5) is a member of the potassium channel subfamily K containing two pore-forming P domains. While the protein has not been definitively shown to form a functional channel independently, current research suggests it may require interaction with non-pore-forming proteins for activity . Its significance lies in understanding potassium channel regulation and function, particularly in neurological and cellular homeostasis contexts.

The protein is encoded by the KCNK15 gene and is also known by several synonyms including K2p15.1, KCNK11, KCNK14, KT3.3, and TASK-5 . Research into KCNK15 contributes to our broader understanding of membrane transport processes and ion channel biology.

Which applications are most commonly used with KCNK15 antibodies?

Based on current antibody availability, KCNK15 antibodies can be utilized in multiple research applications:

The most consistent results are typically observed with Western blot and immunohistochemistry applications, which are supported by multiple antibody vendors and validation studies .

How should I select the appropriate KCNK15 antibody for my experiment?

Selection should be based on multiple factors:

• Target region: Consider whether you need an antibody targeting the N-terminus , C-terminus , middle region , or extracellular domain based on your experimental design.

• Species reactivity: Verify that the antibody recognizes KCNK15 in your model organism. Available antibodies show reactivity with:

  • Human (most common)

  • Mouse

  • Rat

  • Additional species including cow, dog, and pig for some antibodies

• Application compatibility: Confirm the antibody is validated for your specific application. For example, if using multiple methods (WB and IHC), select an antibody validated for both .

• Validation data: Review available validation information, particularly Western blot images or immunohistochemistry results that demonstrate specificity .

What are the best practices for validating KCNK15 antibody specificity?

Rigorous validation is critical due to potential cross-reactivity with other potassium channel family members:

• Pre-adsorption/blocking peptide control: Use corresponding blocking peptides such as KCNK15/TASK-5 (extracellular) Blocking Peptide (e.g., BLP-PC158) to confirm specificity. This allows direct comparison between antibody alone and antibody pre-incubated with the blocking peptide .

• Recommended protocol for blocking peptide validation:

  • Prepare two identical Western blot membranes with target samples

  • Incubate one with anti-KCNK15 antibody (1:200-1:500 dilution)

  • Incubate the second with the same antibody pre-incubated with blocking peptide (1:1 ratio, 1μg peptide per 1μg antibody)

  • Compare signal reduction or elimination in the blocked sample

• Knockout/knockdown controls: If available, include KCNK15 knockout/knockdown samples to validate specificity.

• Multiple antibody approach: Use antibodies targeting different epitopes of KCNK15 to confirm consistent detection patterns .

What are the critical factors for optimizing Western blot protocols with KCNK15 antibodies?

When performing Western blot with KCNK15 antibodies, consider these optimization factors:

• Sample preparation:

  • For tissue samples: Complete lysis buffers containing phosphatase and protease inhibitors are essential

  • Mouse and rat brain lysates have been validated as positive controls

  • Expected molecular weight: approximately 36kDa

• Dilution optimization:

  • Start with manufacturer's recommended range (typically 1:500-1:3000)

  • Perform dilution series to determine optimal signal-to-noise ratio

  • Higher concentrations may be needed for tissues with lower KCNK15 expression

• Blocking conditions:

  • 5% non-fat dry milk in TBST is typically effective

  • For phospho-specific detection, BSA may be preferred over milk proteins

• Detection systems:

  • Enhanced chemiluminescence (ECL) systems provide adequate sensitivity

  • For low-abundance samples, consider super-signal enhanced chemiluminescence reagents

How should immunohistochemistry protocols be modified for optimal KCNK15 detection?

Successful IHC with KCNK15 antibodies requires attention to these parameters:

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval methods are generally preferred

  • Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be tested to determine optimal conditions

• Dilution ranges:

  • For paraffin-embedded sections: 1:50-1:500 depending on the specific antibody

  • For frozen sections: Start with more dilute solutions (1:100-1:500)

• Detection sensitivity:

  • Amplification systems (e.g., tyramide signal amplification) may improve detection of low-abundance KCNK15

  • Polymer detection systems reduce background compared to ABC methods for many KCNK15 antibodies

• Controls:

  • Positive control tissues should be run in parallel

  • Negative controls should include primary antibody omission and ideally blocking peptide competition

What are common problems with KCNK15 antibodies and how can they be addressed?

For persistent issues with Western blot detection, consider:

• Using fresh lysates to minimize protein degradation

• Testing different membrane types (PVDF vs. nitrocellulose)

• Extending antibody incubation times (overnight at 4°C)

• Adding 0.05% SDS to antibody dilution buffer to increase accessibility of epitopes

How can KCNK15 antibodies be effectively used in multiplexed detection systems?

For co-localization or multiple protein detection experiments:

• Fluorophore selection for direct conjugates:

  • KCNK15 antibodies can be conjugated to various fluorophores including AF488, AF555, AF647, and PE

  • When selecting conjugates, consider spectral overlap and microscope filter sets

  • Custom conjugation services are available from several providers

• Protocol considerations for multiplexing:

  • Sequential staining may be required to minimize cross-reactivity

  • For multiple rabbit-derived antibodies, use specialized multiplexing kits that utilize unique labeling strategies

  • Test for cross-reactivity between secondary detection systems

• Flow cytometry applications:

  • PE-conjugated KCNK15 antibodies can be excited at 488-561nm with emission captured at 586/14nm

  • Ensure adequate controls including FMO (fluorescence minus one) controls

What are the latest research applications for KCNK15 antibodies in neuroscience and cancer research?

Advanced research applications include:

• Neuroscience applications:

  • Investigation of KCNK15 expression in neuronal subtypes

  • Co-localization studies with other ion channels and neuronal markers

  • Evaluation of KCNK15 in neurological disorders

• Cancer research:

  • KCNK15 expression has been studied in testis cancer, though it was not found to be prognostic

  • Potential role in tumor cell membrane potential regulation

  • Expression correlation with cancer progression and therapeutic response

• Methodological approaches:

  • Single-cell expression profiling combined with immunofluorescence

  • Proximity ligation assays to detect KCNK15 interactions with other proteins

  • CRISPR-based functional genomics to correlate KCNK15 expression with channel function

How does KCNK15 antibody selection differ for in vivo applications versus in vitro systems?

For in vivo applications (e.g., intracerebroventricular antibody delivery or retro-orbital injection):

• Considerations for in vivo use:

  • Antibody specificity becomes even more critical

  • Sodium azide-free formulations must be used

  • Blood-brain barrier penetration must be considered for CNS applications

  • Antibody stability in physiological conditions should be evaluated

• Technical differences from in vitro applications:

  • Higher antibody concentrations are typically required

  • Bioavailability and tissue distribution should be determined

  • Potential immune responses against antibodies must be monitored

• Controls for in vivo experiments:

  • Include isotype control antibodies (e.g., Rabbit IgG)

  • Validate tissue penetration using labeled antibodies

  • Include target knockout animals when available

What are the key considerations when using KCNK15 antibodies for quantitative protein analysis?

For quantitative applications such as protein expression level assessment:

• Quantification methodologies:

  • Western blot: Use appropriate normalization controls (β-actin, GAPDH)

  • ELISA: Develop standard curves using recombinant KCNK15 protein

  • Immunofluorescence: Use calibrated fluorescence standards

• Technical validation requirements:

  • Linear range determination for each antibody and application

  • Reproducibility assessment across multiple experiments

  • Inter-laboratory validation when possible

• Data analysis approaches:

  • Densitometry for Western blot quantification

  • Standardized scoring systems for immunohistochemistry

  • Automated image analysis for immunofluorescence quantification

How can researchers navigate contradictory results when using different KCNK15 antibodies?

When faced with contradictory results:

• Systematic validation approach:

  • Compare antibody epitope regions (N-terminal vs. C-terminal vs. middle region )

  • Verify species cross-reactivity claims

  • Evaluate detection methods and sensitivity differences

• Reconciliation strategies:

  • Use orthogonal techniques (e.g., mass spectrometry) to confirm protein identity

  • Employ genetic approaches (siRNA, CRISPR) to validate antibody specificity

  • Consider isoform specificity and post-translational modifications

• Reporting contradictory results:

  • Document all experimental variables systematically

  • Report antibody catalog numbers, lot numbers, and dilutions used

  • Consider multiple antibody approaches with clear documentation of similarities and differences

What criteria should guide selection between monoclonal and polyclonal KCNK15 antibodies?

While most currently available KCNK15 antibodies are polyclonal , understanding the trade-offs is important:

For most KCNK15 research, polyclonal antibodies provide adequate performance across multiple applications, particularly for Western blot and immunohistochemistry .

How should researchers design experiments to comprehensively validate KCNK15 antibody performance?

A systematic validation workflow includes:

• Initial characterization:

  • Western blot with positive control tissues (brain lysates)

  • Evaluation of band pattern and molecular weight (~36kDa)

  • Blocking peptide competition studies

• Cross-application validation:

  • Compare antibody performance across intended applications

  • Ensure consistent protein detection patterns between techniques

  • Document application-specific optimization parameters

• Experimental controls integration:

  • Negative controls (primary antibody omission)

  • Blocking peptide controls

  • Isotype controls

  • Genetic validation (knockdown/knockout)

• Documentation recommendations:

  • Maintain detailed records of antibody source, lot, and protocols

  • Image complete blots/gels including molecular weight markers

  • Document all optimization steps and parameter variations