KCNA7 Antibody

What is KCNA7 and what biological role does it serve?

KCNA7 encodes the Kv1.7 voltage-gated potassium channel, a member of the Shaker family of K+ channels. The protein features six membrane-spanning domains with intracellular N- and C-termini. Functionally, Kv1.7 plays a critical role in the repolarization of cell membranes, particularly in cardiac tissue. The biophysical and pharmacological properties of Kv1.7 closely resemble the ultra-rapidly activating delayed rectifier (IKur) in cardiac tissue, which is central to cardiac atrial repolarization. Research suggests that the IKur current may result from heteromeric Kv1.5/Kv1.7 channels. Additionally, Kv1.7 contributes to the cardiac transient outward potassium current (Ito1), a major current responsible for the repolarizing phase 1 of the cardiac action potential .

What tissue expression pattern does KCNA7 exhibit?

Expression analysis reveals that KCNA7 mRNA is predominantly expressed in the heart, with significant levels also detected in skeletal muscle and pancreas. Lower expression levels are found in kidney, brain, and pancreatic islet cells. Some studies also report minimal expression in liver and other tissues. Northern blot analysis has identified a single mRNA isoform of approximately 4.5 kb .

What types of KCNA7 antibodies are commercially available?

Multiple types of KCNA7 antibodies are available for research applications, including:

• Antibody formats: Both monoclonal and polyclonal antibodies

• Host species: Primarily rabbit and mouse-derived antibodies

• Target species reactivity: Antibodies that recognize human, mouse, rat, and sometimes other species

• Applications compatibility: Antibodies validated for Western blot (WB), ELISA, immunohistochemistry (IHC), and immunocytochemistry (ICC)

• Conjugations: Available as unconjugated antibodies or conjugated to various fluorophores (e.g., FL594, FITC) or enzymes (e.g., HRP)

How should I select the appropriate KCNA7 antibody for my specific application?

Selection of an appropriate KCNA7 antibody should be based on:

• Target species compatibility: Ensure the antibody recognizes KCNA7 in your species of interest. For example, anti-mouse Kv1.7 antibody (#APC-063) is specifically designed for mouse samples and will not recognize human or rat KCNA7 .

• Application requirements: Different antibodies are optimized for specific applications:

  • For protein quantification: Select antibodies validated for Western blot

  • For localization studies: Choose antibodies validated for IHC or ICC

  • For protein-protein interaction studies: Consider antibodies validated for immunoprecipitation

• Epitope targeting: Consider the structural region targeted by the antibody:

  • N-terminal antibodies (e.g., targeting amino acids 2-15 of mouse Kv1.7)

  • C-terminal antibodies (e.g., targeting the C-terminal region of mouse Kv1.7)

  • Fusion protein-derived antibodies (e.g., targeting amino acids 1-44 and 450-480)

• Validation evidence: Review validation data available from manufacturers, including Western blot images showing expected molecular weight (~50 kDa) or immunostaining patterns .

What are the recommended protocols for using KCNA7 antibodies in Western blot applications?

For optimal results in Western blot applications using KCNA7 antibodies:

• Sample preparation:

  • For tissue samples: Use membrane fractions from heart, skeletal muscle, or other KCNA7-expressing tissues

  • For cell lysates: Consider using cells known to express KCNA7 or overexpression systems

• Antibody dilution ranges:

  • Typical working dilutions range from 1:200 to 1:2000 depending on the specific antibody

  • For example, Anti-Mouse Kv1.7 antibody (#APC-063) is recommended at 1:200 dilution

  • Other antibodies like orb524383 are recommended at 1:500-1:2000 for Western blot

• Blocking and incubation conditions:

  • Use appropriate blocking buffer (typically 5% non-fat milk or BSA)

  • Incubate primary antibody overnight at 4°C for optimal results

  • Follow with appropriate secondary antibody detection system

• Controls:

  • Positive control: Use tissue known to express KCNA7 (e.g., heart membranes)

  • Negative control: Include antibody preincubation with blocking peptide when available

  • Expected molecular weight: ~50 kDa band for KCNA7

How can I optimize immunohistochemistry protocols for KCNA7 detection?

For successful immunohistochemical detection of KCNA7:

• Tissue preparation:

  • Use freshly fixed tissues (paraffin-embedded or frozen sections)

  • Consider tissue-specific expression patterns (heart, skeletal muscle, pancreas, kidney)

• Antibody dilutions:

  • For IHC-P applications, typical dilutions range from 1:50 to 1:200

  • For immunofluorescence labeling, 1:100 dilution is often recommended

• Antigen retrieval:

  • May be necessary for paraffin-embedded tissues

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Detection systems:

  • For brightfield microscopy: HRP-conjugated secondary antibodies with DAB

  • For fluorescence microscopy: Appropriate fluorophore-conjugated secondary antibodies or directly conjugated primary antibodies (e.g., FL594-conjugated anti-Kv1.7)

• Controls:

  • Positive control: heart or skeletal muscle tissue sections

  • Negative control: tissues with low/no KCNA7 expression or primary antibody omission

How can KCNA7 antibodies be used to investigate heteromeric channel formation?

To investigate potential heteromeric channel formation between KCNA7 and other Kv channels:

• Co-immunoprecipitation (Co-IP):

  • Use KCNA7 antibodies to immunoprecipitate the protein complex

  • Probe for potential interaction partners (e.g., Kv1.5) by Western blot

  • Consider reciprocal Co-IP to confirm interactions

• Proximity ligation assay (PLA):

  • Utilize KCNA7 antibodies alongside antibodies against potential interaction partners

  • This technique allows visualization of protein-protein interactions (<40 nm) in situ

• Double immunofluorescence labeling:

  • Use FL594-conjugated anti-Kv1.7 antibody alongside differently labeled antibodies against potential partners

  • Analyze colocalization using confocal microscopy and quantitative colocalization analysis

• Experimental controls:

  • Positive controls: Known interaction partners

  • Negative controls: Proteins not expected to interact with KCNA7

  • Validation: Functional assays to confirm physiological relevance of interactions

The research data suggests that Kv1.7 may form heteromers with other Shaker subfamily members, particularly with Kv1.5 to generate the IKur current in cardiac tissue . Investigating these interactions could provide valuable insights into cardiac electrophysiology.

What approaches can be used to correlate KCNA7 expression with cardiac electrophysiological phenotypes?

To establish correlations between KCNA7 expression and cardiac function:

• Quantitative expression analysis:

  • Use validated KCNA7 antibodies for Western blot quantification

  • Correlate protein levels with electrophysiological measurements

  • Compare expression across different cardiac regions or disease models

• Functional electrophysiology:

  • Patch-clamp recordings from isolated cardiomyocytes

  • Correlation of IKur current properties with KCNA7 expression levels

  • Pharmacological manipulation with specific Kv channel modulators

• Genetic manipulation approaches:

  • siRNA knockdown of KCNA7 followed by antibody-based validation of knockdown efficiency

  • Correlation of knockdown efficiency with functional changes

  • Rescue experiments with wild-type or mutant KCNA7

• Pathological correlations:

  • Compare KCNA7 expression between normal and diseased cardiac tissue using antibody-based methods

  • Correlate expression changes with arrhythmia susceptibility or other cardiac phenotypes

This approach would help determine the specific contribution of Kv1.7 to cardiac repolarization currents, which has been suggested to involve both the ultra-rapidly activating delayed rectifier (IKur) and the transient outward potassium current (Ito1) .

What strategies should be employed when facing non-specific binding with KCNA7 antibodies?

When encountering non-specific binding issues:

• Antibody validation:

  • Use blocking peptides when available (e.g., Mouse Kv1.7/KCNA7 Blocking Peptide #BLP-PC063)

  • Test multiple antibodies targeting different epitopes

  • Include appropriate negative controls (tissues/cells not expressing KCNA7)

• Protocol optimization:

  Optimization Parameter	Standard Condition	Suggested Modifications
  Blocking reagent	5% BSA or milk	Try alternative blockers (e.g., serum, commercial blockers)
  Antibody dilution	As recommended	Increase dilution (e.g., from 1:200 to 1:500)
  Incubation time	Overnight at 4°C	Reduce to 2-4 hours at room temperature
  Washing steps	3 × 5 min	Increase to 5 × 5 min with higher salt concentration
  Secondary antibody	As recommended	More extensively cross-adsorbed secondaries

• Sample preparation improvements:

  • Optimize protein extraction protocols for membrane proteins

  • Consider membrane fractionation to enrich for KCNA7

  • Use fresh samples and avoid freeze-thaw cycles

• Alternative detection methods:

  • If Western blot shows non-specific bands, try immunoprecipitation followed by Western blot

  • For IHC/ICC, consider antigen retrieval optimization or fluorescence detection

How can I reconcile conflicting data between antibody-based detection and functional measurements of KCNA7?

When antibody-based data conflicts with functional data:

• Multiple antibody approach:

  • Use antibodies targeting different epitopes (N-terminal vs. C-terminal)

  • Compare monoclonal vs. polyclonal antibodies

  • Validate with genetic approaches (siRNA, CRISPR/Cas9)

• Post-translational modifications:

  • Consider that antibody detection might be affected by phosphorylation or other modifications

  • Use phospho-specific antibodies if available

  • Treat samples with phosphatases prior to antibody detection

• Splice variant considerations:

  • Investigate potential splice variants that might not be detected by certain antibodies

  • Compare with mRNA expression data (e.g., the 4.5 kb transcript reported)

  • Consider species differences (e.g., N-terminal sequence differences between human and mouse)

• Heteromeric channels:

  • Functional properties might reflect heteromeric channels (e.g., Kv1.5/Kv1.7)

  • Use co-immunoprecipitation to identify channel composition

  • Correlate antibody-detected expression with specific current components

• Quantitative correlation analysis:

  • Plot antibody-detected protein levels against functional measurements

  • Analyze for non-linear relationships or threshold effects

  • Consider time-dependent or activity-dependent regulation

This analytical approach acknowledges that KCNA7 may form heteromeric channels with other Kv family members, which could affect both antibody detection and functional properties .

What considerations are important when comparing KCNA7 expression across different species?

When conducting cross-species comparisons:

• Antibody cross-reactivity considerations:

  • Some antibodies are species-specific (e.g., Anti-Mouse Kv1.7 antibody does not recognize human or rat KCNA7)

  • Others have broader cross-reactivity (e.g., antibodies recognizing human, mouse, and rat)

  • Verify species reactivity before comparative studies

• Sequence homology analysis:

  • Human KCNA7 protein shares 91% amino acid identity with mouse Kcna7

  • C-terminal regions may have higher conservation than N-terminal regions

  • Some peptide immunogens show species variations (e.g., 85.7% homology between mouse and human sequences)

• Expression pattern differences:

  • Expression levels may vary between species in the same tissue

  • Compare relative expression patterns rather than absolute levels

  • Validate with species-specific positive controls

• Functional correlation approach:

  • Correlate antibody detection with electrophysiological measurements in each species

  • Compare the contribution of KCNA7 to specific currents across species

  • Consider evolutionary conservation of channel function versus expression pattern

This approach acknowledges the significant homology between species (91% between human and mouse), while recognizing potential differences that may affect antibody binding and experimental interpretation .