KCNA7 Antibody, HRP conjugated

What is KCNA7 and what is its biological significance?

KCNA7 (potassium voltage-gated channel subfamily A member 7) is a member of the voltage-gated potassium channel family that mediates the voltage-dependent potassium ion permeability of excitable membranes. It assumes opened or closed conformations in response to voltage differences across the membrane, forming potassium-selective channels through which potassium ions may pass in accordance with their electrochemical gradient .

KCNA7 belongs to the shaker-related subfamily of potassium channels. Unlike other Kv1-family genes that contain intronless coding regions, KCNA7 is encoded by two exons separated by a conserved intron . This structural distinction makes it unique among the potassium channel family.

The protein plays crucial roles in:

• Regulating neurotransmitter release

• Controlling neuronal excitability

• Mediating electrolyte transport

• Contributing to muscle contraction and cell volume regulation

What are the structural characteristics of HRP-conjugated antibodies against KCNA7?

HRP-conjugated KCNA7 antibodies typically consist of:

• Recognition domain: Polyclonal antibodies raised against specific peptide regions of human KCNA7, commonly using recombinant human potassium voltage-gated channel subfamily A member 7 protein (amino acids 1-143) as the immunogen .

• Host origin: Predominantly raised in rabbits, though other species may be used depending on the manufacturer .

• Conjugation chemistry: Horseradish peroxidase (HRP) is covalently linked to the antibody molecule, typically through controlled chemical conjugation methods that preserve both antibody binding and enzymatic activity.

• Buffer composition: Most commercially available HRP-conjugated KCNA7 antibodies are supplied in a buffer containing:

  • 0.01M PBS, pH 7.4

  • 0.03% Proclin-300 as preservative

  • 50% glycerol for stability

• Purity level: These antibodies are typically purified using Protein G chromatography, achieving >95% purity .

What are the optimal applications for KCNA7 Antibody, HRP conjugated in research settings?

• ELISA: The primary application with recommended dilutions between 1:2000-1:10000 . This is particularly valuable for quantitative measurements of KCNA7 protein levels in tissue or cell lysates.

• Western Blotting: While not all HRP-conjugated KCNA7 antibodies are validated for western blotting, those that are typically require dilutions of approximately 1:500 to 1:1000 . The expected molecular weight for KCNA7 is approximately 50 kDa .

• Immunohistochemistry: Some KCNA7 antibodies show good performance in IHC applications at dilutions of 1:50-1:200, particularly in human tissues such as liver cancer and colon cancer samples .

When using these antibodies, researchers should:

• Validate specificity using appropriate controls

• Optimize dilutions for their specific experimental system

• Consider the buffer conditions that may affect antibody performance

How should experimental controls be designed when using KCNA7 Antibody, HRP conjugated?

Proper experimental controls are critical for interpreting results with KCNA7 Antibody, HRP conjugated:

• Positive tissue controls:

  • Human colon cancer and liver cancer tissues have been verified as appropriate positive controls for KCNA7 antibodies .

  • These tissues demonstrate consistent KCNA7 expression patterns that can validate antibody specificity.

• Negative controls:

  • Peptide blocking: Pre-incubating the antibody with the immunizing peptide should abolish specific staining .

  • Isotype controls: Rabbit IgG at the same concentration as the primary antibody can help distinguish non-specific binding.

  • Tissues known to lack KCNA7 expression.

• Technical controls:

  • Secondary antibody-only controls to assess background.

  • Endogenous peroxidase blocking verification.

  • Gradient dilution series to determine optimal antibody concentration.

• Biological validation:

  • Compare results with gene expression data from RT-PCR.

  • Consider using tissues from different species to verify cross-reactivity claims.

For example, in immunohistochemistry applications, researchers have demonstrated that KCNA7 antibody staining at 1:40 dilution shows specific staining in human liver cancer and colon cancer tissues that can be abolished by treatment with the synthetic peptide used for immunization .

What are the optimal storage and handling conditions for maintaining KCNA7 Antibody, HRP conjugated activity?

To preserve the activity and specificity of KCNA7 Antibody, HRP conjugated, follow these evidence-based storage and handling guidelines:

• Long-term storage:

  • Store at -20°C in small aliquots to prevent freeze-thaw cycles .

  • Avoid repeated freezing and thawing, which can degrade both the antibody and the HRP enzyme.

• Short-term storage:

  • For usage within 2 weeks, refrigeration at 2-8°C is acceptable .

  • Keep protected from light, as HRP is photosensitive.

• Working dilutions:

  • Prepare fresh dilutions on the day of use.

  • Use buffers containing 1-5% BSA or non-fat dry milk as blockers to reduce non-specific binding.

  • For Western blot applications, consider adding 0.1% Tween-20 to reduce background.

• Handling precautions:

  • Centrifuge the vial prior to opening to ensure collection of all material.

  • Avoid microbial contamination which can degrade the antibody and introduce proteases.

  • Do not expose to strong oxidizing agents that can affect HRP activity.

The shelf life under optimal storage conditions is typically 12-24 months from the date of receipt .

How should researchers optimize Western blot protocols specifically for KCNA7 Antibody, HRP conjugated?

While some KCNA7 Antibody, HRP conjugated preparations are primarily validated for ELISA, researchers may adapt them for Western blot applications by following this optimized protocol derived from literature on similar antibodies:

Sample preparation:

• Extract proteins using N-PER Protein Extraction Reagent supplemented with protease inhibitor cocktail .

• Determine protein concentration using BCA or Bradford assay.

• Denature samples in Laemmli buffer (with β-mercaptoethanol) at 95°C for 5 minutes.

Gel electrophoresis and transfer:

• Load 20-50 μg of protein per lane on a 4-12% Bis-Tris gel.

• Run at constant 125V for approximately 3 hours for optimal separation .

• Transfer to PVDF membrane (preferable over nitrocellulose for potassium channel proteins).

Antibody incubation:

• Block membrane in TBS-T with 5% non-fat milk for 1.5 hours at room temperature .

• Incubate with KCNA7 Antibody, HRP conjugated at 1:500-1:1000 dilution in blocking buffer overnight at 4°C.

• Wash three times (25 minutes each) with TBS-T .

Detection:

• Since the antibody is HRP-conjugated, proceed directly to chemiluminescent detection.

• Use SuperSignal West Atto Ultimate Sensitivity ECL reagents for optimal detection .

• Expose for 30 seconds initially, then adjust exposure time as needed.

Troubleshooting steps:

• If high background is observed, increase washing time and detergent concentration.

• If signal is weak, consider using a signal enhancer compatible with HRP.

• The expected molecular weight for KCNA7 is ~50 kDa , but post-translational modifications may result in higher apparent molecular weights.

How can KCNA7 Antibody, HRP conjugated be utilized in studying neurological disorders?

KCNA7 has been implicated in several neurological conditions, and HRP-conjugated antibodies can be valuable tools for investigating these associations:

• Neurodevelopmental disorders:
  Research has identified KCNA7 as differentially expressed in neurons derived from patients with certain neurodevelopmental syndromes. For example, comparative studies of neurons from ROHHAD (Rapid-onset Obesity with Hypothalamic dysfunction, Hypoventilation, and Autonomic Dysregulation), CCHS (Congenital Central Hypoventilation Syndrome), and PWS (Prader-Willi Syndrome) patients revealed transcriptional changes in potassium channel genes .

• Methodological approach:

  • Isolate neuronal cultures from patient-derived dental pulp stem cells (DPSC)

  • Perform protein extraction using N-PER with protease inhibitors

  • Use KCNA7 Antibody, HRP conjugated in Western blot analysis at 1:500 dilution

  • Normalize expression to GAPDH (1:10,000 dilution)

  • Quantify using ImageJ Fiji gel quantification plugin

• Spatial expression patterns:

  • Use KCNA7 Antibody, HRP conjugated in immunohistochemistry at 1:50-1:200 dilution on brain tissue sections

  • Compare expression patterns between affected and control tissues

  • Validate findings with RNA expression data from the same regions

• Co-localization studies:

  • Dual staining with markers of neuronal subtypes

  • Analysis of subcellular localization in relation to synaptic markers

  • Assessment of KCNA7 distribution in relation to other ion channels

This approach has revealed that KCNA7 expression changes may contribute to the neuronal dysfunction observed in certain disorders, particularly those affecting autonomic nervous system regulation .

What are the most effective approaches for using KCNA7 Antibody, HRP conjugated in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) with KCNA7 Antibody, HRP conjugated presents unique challenges but can be effectively implemented using this optimized protocol based on literature for similar ion channel antibodies:

Pre-clearing the lysate:

• Prepare cell/tissue lysate in a non-denaturing buffer containing:

  • 150 mM NaCl

  • 20 mM Tris-HCl (pH 7.4)

  • 1% Triton X-100

  • 1 mM EDTA

  • Protease inhibitor cocktail

• Pre-clear by incubating with Protein A/G beads for 1 hour at 4°C.

• Remove beads by centrifugation (14,000g for 10 minutes).

Immunoprecipitation:

• For direct IP (not recommended with HRP-conjugated antibodies):

  • The presence of HRP can interfere with subsequent antibody detection steps.

  • Instead, use unconjugated KCNA7 antibody at 1:160 dilution.

  • Incubate with lysate for 45 minutes on ice .

  • Add Protein A/G beads and rock gently for 1 hour at 4°C .

• For indirect IP (recommended approach):

  • First capture KCNA7 using unconjugated antibody.

  • Detect associated proteins using KCNA7 Antibody, HRP conjugated in Western blot analysis.

• Key controls to include:

  • 5% of total cell lysate as loading control

  • IgG control to identify non-specific binding

  • Reversed Co-IP to confirm interaction

Western blot detection:

• Prepare samples as described in standard western blot protocols.

• Probe with KCNA7 Antibody, HRP conjugated at 1:500 dilution.

• Develop with chemiluminescent substrate suitable for HRP.

This approach has been successfully used to investigate interactions between potassium channels and associated proteins, including other Kv channel subunits like KCNA2, KCNA4, KCNA5, and KCNA6 .

How does the performance of polyclonal KCNA7 Antibody, HRP conjugated compare to monoclonal alternatives in research applications?

When comparing polyclonal KCNA7 Antibody, HRP conjugated to monoclonal alternatives, researchers should consider several performance parameters:

Recommendations based on application:

• For quantitative ELISA: Polyclonal HRP-conjugated antibodies generally provide better sensitivity for detecting KCNA7 in solution.

• For Western blotting: Monoclonal antibodies may provide cleaner results with less background, though polyclonal HRP-conjugated antibodies can be suitable when optimized.

• For immunohistochemistry: The choice depends on the specific research question:

  • Polyclonal antibodies may detect KCNA7 even if partially denatured or modified

  • Monoclonal antibodies provide more consistent staining patterns across experiments

• For co-immunoprecipitation: Monoclonal antibodies are generally preferred due to their higher specificity and lower background.

For most critical applications requiring validation of findings, researchers should consider using both antibody types to confirm results.

What methodologies should be employed to validate the specificity of KCNA7 Antibody, HRP conjugated?

Rigorous validation of KCNA7 Antibody, HRP conjugated specificity is essential for reliable research outcomes. Implement these comprehensive validation approaches:

• Genetic approaches:

  • siRNA/shRNA knockdown: Transfect cells with KCNA7-specific siRNA and confirm reduced antibody signal

  • CRISPR/Cas9 knockout: Generate KCNA7-knockout cell lines as definitive negative controls

  • Overexpression: Transfect cells with KCNA7 expression constructs and verify increased signal intensity

• Biochemical validation:

  • Peptide competition: Pre-incubate antibody with immunizing peptide (synthetic peptide of human KCNA7) before application

  • Multiple antibody comparison: Test parallel detection with antibodies targeting different KCNA7 epitopes

  • Mass spectrometry: Confirm identity of immunoprecipitated proteins detected by the antibody

• Cross-reactivity assessment:

  • Recombinant protein array: Test against related Kv channel proteins (KCNA1-6)

  • Tissue panel screening: Evaluate antibody performance across tissues with known KCNA7 expression patterns

  • Species cross-reactivity: Test on samples from different species to confirm conservation of recognized epitopes

• Correlation with transcript expression:

  • RT-PCR validation: Compare protein detection with KCNA7 mRNA levels

  • RNAseq correlation: Align antibody-based protein quantification with transcriptomic data

Validation case example:
In immunohistochemistry applications, researchers have demonstrated validation by showing that KCNA7 antibody staining at 1:40 dilution produces specific staining in human liver cancer and colon cancer tissues that can be abolished by pre-treatment with the synthetic peptide used for immunization, confirming specificity to the target epitope .

How can KCNA7 Antibody, HRP conjugated be utilized in multiplexed detection systems?

Emerging multiplexed detection systems offer powerful approaches for studying KCNA7 in complex biological contexts. KCNA7 Antibody, HRP conjugated can be integrated into these systems through:

• Sequential multiplexed immunohistochemistry:

  • First detection: Use KCNA7 Antibody, HRP conjugated with DAB substrate (brown precipitate)

  • Stripping/blocking: Inactivate HRP and remove antibody using glycine-SDS buffer (pH 2.0)

  • Subsequent detection: Apply antibodies against other targets with alternative chromogens

  • This method allows visualization of multiple proteins on the same tissue section with spatial context

• Tyramide signal amplification (TSA) multiplexing:

  • KCNA7 Antibody, HRP conjugated catalyzes the deposition of fluorophore-labeled tyramide

  • The covalently bound fluorophore remains after antibody stripping

  • Multiple proteins can be sequentially detected on the same sample

  • This method is particularly valuable for detecting low-abundance proteins like KCNA7

• Multiplex Western blotting strategies:

  • Size-based multiplexing: Detect KCNA7 (~50 kDa) alongside proteins of different molecular weights

  • Fluorescent multiplexing: Combine with antibodies labeled with different fluorophores

  • Strip-and-reprobe: Use KCNA7 Antibody, HRP conjugated first, then strip and reprobe with antibodies against other proteins

• Microfluidic-based approaches:

  • Integrate KCNA7 Antibody, HRP conjugated into microfluidic devices for spatial protein profiling

  • Combine with single-cell analysis techniques for heterogeneity assessment

  • Develop automated, high-throughput KCNA7 detection platforms

These approaches allow researchers to investigate KCNA7 in the context of other ion channels, regulatory proteins, and cell-type markers, providing deeper insights into its functional relationships and regulatory networks.

What are the methodological considerations for using KCNA7 Antibody, HRP conjugated in studying gene expression changes in neurological disorders?

KCNA7 Antibody, HRP conjugated can be a powerful tool for investigating KCNA7 protein expression changes in neurological disorders when integrated with comprehensive methodological approaches:

• Patient-derived cellular models:

  • Dental pulp stem cell (DPSC) approach: Isolate and differentiate patient-derived DPSCs into neuronal cultures for protein analysis

  • iPSC neuronal differentiation: Generate disease-specific neurons from patient fibroblasts

  • Protein extraction protocol: Use N-PER with protease inhibitors, resolve on 4-12% Bis-Tris gels, and transfer to PVDF membrane

  • Detection strategy: KCNA7 Antibody, HRP conjugated (1:500 dilution) with SuperSignal West Atto ECL reagents

• Multi-omics correlation analysis:

  • Integrated approach: Correlate protein expression detected by KCNA7 Antibody, HRP conjugated with:

    • Transcriptomic data from RNAseq (normalized counts)

    • Genomic variations in the KCNA7 gene

    • Epigenetic modifications at the KCNA7 locus

  • Validation strategy: Confirm protein-level changes observed with antibody detection match transcript-level changes

• Disease-specific considerations:
  As shown in recent research, KCNA7 expression changes have been observed in several neurological conditions:

  Disease	Reported KCNA7 Expression Change	Validation Method	Reference
  ROHHAD	Variable expression patterns across patients	Western blot with protein quantification
  CCHS	Reduced expression compared to control	Normalized to GAPDH, quantified with ImageJ
  Familial Atrial Fibrillation	Associated with KCNA7 variants	Western blot analysis of variant proteins

• Methodological controls:

  • Technical controls: Include loading controls (GAPDH at 1:10,000 dilution)

  • Biological controls: Compare age-matched and sex-matched control samples

  • Disease controls: Include samples from related disorders to assess specificity of changes

• Data analysis framework:

  • Normalize KCNA7 expression to housekeeping proteins

  • Perform statistical analysis appropriate for sample size

  • Consider patient heterogeneity in interpretation of results

  • Correlate protein expression with clinical phenotypes when available

This integrated approach allows researchers to establish meaningful connections between KCNA7 expression changes and neurological disease mechanisms, potentially identifying new therapeutic targets or biomarkers.

What are the detailed technical specifications for commercially available KCNA7 Antibody, HRP conjugated products?

The following technical specifications are compiled from multiple commercial sources of KCNA7 Antibody, HRP conjugated products:

Additional Technical Notes:

• These antibodies target Potassium voltage-gated channel subfamily A member 7, also known as Voltage-gated potassium channel subunit Kv1.7

• The KCNA7 gene encodes a protein of 456 amino acid residues

• Unlike other Kv1-family genes with intronless coding regions, KCNA7 is encoded by two exons separated by a conserved intron

• The protein is primarily localized to the membrane

These specifications provide researchers with comprehensive information needed to evaluate the suitability of KCNA7 Antibody, HRP conjugated for their specific experimental applications.

How does the sensitivity and detection limit of KCNA7 Antibody, HRP conjugated compare across different detection systems?

The sensitivity and detection limit of KCNA7 Antibody, HRP conjugated varies significantly depending on the detection system employed. Understanding these differences is critical for experimental design and data interpretation:

Performance factors affecting sensitivity:

• Antibody affinity and specificity: The polyclonal nature of KCNA7 Antibody, HRP conjugated provides recognition of multiple epitopes, potentially enhancing sensitivity but requiring careful optimization to minimize background .

• Antigen accessibility: Membrane proteins like KCNA7 may require optimization of sample preparation:

  • For Western blots: Complete denaturation in SDS buffer is critical

  • For IHC/ICC: Appropriate antigen retrieval methods must be employed

• HRP enzyme activity:

  • Preserve activity by avoiding sodium azide in working solutions

  • Prepare fresh working dilutions

  • Store at recommended temperatures (-20°C for long term)

• Detection reagent quality:

  • Use high-sensitivity ECL reagents for Western blotting

  • For colorimetric detection, use premium-grade DAB substrates

  • Consider signal enhancers for low-abundance targets