KCNJ5 Antibody, FITC conjugated

What is KCNJ5 and why is it an important research target?

KCNJ5 (Potassium Inwardly-Rectifying Channel, Subfamily J, Member 5) is a G protein-activated inward rectifier potassium channel that plays critical roles in cardiac electrophysiology and aldosterone secretion regulation. This membrane protein is characterized by a greater tendency to allow potassium to flow into the cell rather than out of it, with voltage dependence regulated by extracellular potassium concentration . KCNJ5 is known by several synonyms including Cardiac inward rectifier (CIR), G protein-activated inward rectifier potassium channel 4 (GIRK4), Heart KATP channel, Inward rectifier K(+) channel Kir3.4, and IRK-4 . Research interest in KCNJ5 has intensified due to its implications in primary aldosteronism and cardiac arrhythmias, making antibodies against this protein valuable tools for investigating channel localization, expression patterns, and protein interactions in physiological and pathological states.

What are the key specifications of commercially available KCNJ5 Antibody, FITC conjugated?

KCNJ5 Antibody, FITC conjugated, is typically a rabbit polyclonal antibody that targets specific amino acid regions of the KCNJ5 protein. Based on available commercial products, these antibodies generally target the C-terminal region, with specific binding to amino acid regions 348-419 or 350-450 of the human GIRK4 protein . They are produced at high purity (>95%, Protein G purified) and maintained at concentrations of approximately 0.53-0.57 μg/μl in antibody stabilization buffer . The FITC (fluorescein isothiocyanate) conjugation provides fluorescent properties with excitation at 490nm and emission at 525nm, making these antibodies directly applicable for immunofluorescence techniques without secondary antibody requirements .

What experimental applications are supported by KCNJ5 Antibody, FITC conjugated?

KCNJ5 Antibody, FITC conjugated supports multiple experimental applications crucial for ion channel research. These applications include:

This versatility makes FITC-conjugated KCNJ5 antibodies particularly valuable for multidimensional research approaches investigating channel expression, localization, and interaction dynamics in various experimental contexts .

What species reactivity can be expected with KCNJ5 Antibody, FITC conjugated?

Different commercially available KCNJ5 Antibody, FITC conjugated preparations demonstrate varied species reactivity profiles. Some antibodies are specifically human-reactive , while others show broader cross-reactivity with human, mouse, and rat KCNJ5 proteins . This cross-reactivity is particularly valuable for comparative studies across species models. For researchers working with less common experimental models, it's important to note that certain antibody preparations may also show reactivity with rabbit and bat KCNJ5, though these are less commonly available in FITC-conjugated formats . Species reactivity is determined by the conservation of the epitope sequence across species and should be carefully considered when designing experiments, particularly for evolutionary or comparative physiology studies.

What are the recommended storage and handling conditions for maintaining KCNJ5 Antibody, FITC conjugated activity?

Proper storage and handling of KCNJ5 Antibody, FITC conjugated is critical for maintaining its immunoreactivity and fluorescence properties. These antibodies should be stored at -20°C for long-term preservation of activity . It's important to note that FITC is light-sensitive, so exposure to light should be minimized during all handling procedures to prevent photobleaching. Repeated freeze-thaw cycles should be avoided, so aliquoting the antibody upon first thaw is recommended for laboratories planning multiple experiments over time. When working with the antibody, maintain cold conditions (on ice) and use appropriate stabilization buffers as indicated by the manufacturer. For diluted working solutions, prepare them fresh before each experiment and store remaining stock solution protected from light. Following these precautions will help ensure consistent experimental results and maximize the usable lifetime of these specialized research reagents.

How can KCNJ5 Antibody, FITC conjugated be optimized for co-localization studies with other channel proteins?

For co-localization studies investigating KCNJ5's interaction with other ion channels or regulatory proteins, researchers need to carefully design their experimental approach to leverage the FITC conjugation while enabling multiple protein detection. The FITC conjugate on KCNJ5 antibodies has excitation at 490nm and emission at 525nm (green fluorescence) , which constrains the selection of other fluorophores to avoid spectral overlap. For optimal co-localization studies:

• Select complementary fluorophores for other target proteins with minimal spectral overlap with FITC (e.g., Cy3, Cy5, or Alexa 647)

• Implement sequential scanning protocols when using confocal microscopy to minimize cross-channel bleed-through

• Include appropriate negative controls and single-stained samples for accurate spectral unmixing

• Consider photobleaching characteristics during experimental design, as FITC may bleach more rapidly than other fluorophores

• When investigating membrane protein interactions, super-resolution techniques such as STORM or PALM may provide enhanced spatial resolution beyond the diffraction limit

When optimized, these approaches allow researchers to study KCNJ5's spatial relationship with regulatory proteins or other potassium channel family members in native tissue or heterologous expression systems.

What methodological approaches can resolve contradictory KCNJ5 localization data between Western blot and immunofluorescence studies?

Discrepancies between Western blot and immunofluorescence results for KCNJ5 localization are not uncommon and can arise from several methodological factors. To resolve such contradictions, consider implementing these methodological approaches:

• Epitope accessibility verification: The folding of KCNJ5 in fixed tissues may differ from denatured proteins in Western blots. Use multiple antibodies targeting different KCNJ5 epitopes (N-terminal, C-terminal) to confirm localization.

• Fixation optimization: Test multiple fixation protocols (PFA, methanol, acetone) as different fixatives may alter epitope exposure differently.

• Permeabilization assessment: For membrane proteins like KCNJ5, permeabilization conditions critically affect antibody access. Systematically test different detergents (Triton X-100, saponin) and concentrations.

• Subcellular fractionation validation: Perform subcellular fractionation followed by Western blotting to biochemically verify localization patterns observed in immunofluorescence.

• Genetic validation: Use CRISPR/Cas9 to create epitope-tagged KCNJ5 or knockout controls to validate antibody specificity.

By systematically exploring these variables, researchers can identify the source of discrepancies and establish reliable protocols for consistent KCNJ5 detection across multiple experimental platforms.

How can KCNJ5 Antibody, FITC conjugated be effectively utilized in live-cell imaging experiments?

While FITC-conjugated antibodies are traditionally used for fixed samples, advances in membrane-permeant antibody delivery methods have opened possibilities for live-cell applications with KCNJ5 Antibody, FITC conjugated. For effective live-cell imaging:

• Antibody delivery optimization: Test protein transfection reagents (Chariot, BioPORTER) that can deliver functional antibodies into live cells while maintaining their binding properties.

• Microinjection approach: For single-cell studies, precision microinjection of diluted antibody (1:500-1:1000) can provide controlled intracellular delivery while minimizing cellular stress.

• Cell membrane permeabilization: Gentle permeabilization using Streptolysin O (SLO) at low concentrations allows temporary antibody access while maintaining cell viability.

• pH considerations: Since FITC fluorescence is pH-sensitive, buffer your imaging media to pH 7.4 and monitor for intracellular pH changes that might affect signal intensity.

• Phototoxicity mitigation: Minimize exposure times and light intensity, and include antioxidants in imaging media to reduce phototoxicity from FITC excitation.

• Temporal resolution: Acquire images at appropriate intervals to capture KCNJ5 trafficking dynamics while minimizing photobleaching.

These approaches enable the study of KCNJ5 dynamics in physiologically relevant conditions, though researchers should validate that antibody binding doesn't disrupt channel function through electrophysiological controls.

What are the critical considerations for quantitative analysis of KCNJ5 expression using FITC-conjugated antibodies in flow cytometry?

Flow cytometry offers unique advantages for quantitative analysis of KCNJ5 expression across cell populations. When using KCNJ5 Antibody, FITC conjugated for flow cytometry, consider these critical factors:

• Sample preparation optimization:

  • For membrane proteins like KCNJ5, gentle fixation (0.5-2% paraformaldehyde) preserves epitope accessibility

  • Permeabilization conditions must be optimized for intracellular epitopes vs. membrane-exposed regions

• Antibody titration:

  • Determine optimal antibody concentration through systematic titration (typically starting at 1:100-1:500)

  • Establish signal-to-noise ratio by comparing to isotype controls and unstained samples

• Fluorescence compensation:

  • FITC signal (525nm) may overlap with other fluorophores in multiplex experiments

  • Use single-stained controls for each fluorophore to establish proper compensation matrices

• Quantitative calibration:

  • Use quantitative fluorescence calibration beads to convert arbitrary fluorescence units to antibodies bound per cell

  • This enables absolute quantification of KCNJ5 expression levels across different experimental conditions

• Controls for specific detection:

  • Include cells with confirmed KCNJ5 knockout or knockdown

  • Use blocking peptides specific to the antibody's epitope region (348-419 or 350-450) to confirm binding specificity

By addressing these considerations, researchers can achieve reliable quantitative assessment of KCNJ5 expression levels in heterogeneous cell populations and correlate expression with functional or pathological states.

How can researchers validate the specificity of KCNJ5 Antibody, FITC conjugated for their particular experimental system?

Validating antibody specificity is essential for producing reliable scientific results, particularly for ion channel research where protein families share structural similarities. For KCNJ5 Antibody, FITC conjugated, implement these validation strategies:

• Genetic validation approaches:

  • KCNJ5 knockdown/knockout verification: Compare antibody staining in wild-type versus KCNJ5-depleted samples

  • Heterologous expression: Test antibody in expression systems (HEK293) with and without KCNJ5 transfection

  • Epitope-tagged constructs: Create KCNJ5 with alternative tags (HA, Flag) and confirm co-localization

• Biochemical validation:

  • Peptide competition: Pre-incubate antibody with immunizing peptide (from regions 348-419 or 350-450) to block specific binding

  • Mass spectrometry: Perform immunoprecipitation followed by MS to confirm target identity

  • Orthogonal antibodies: Compare staining patterns with antibodies targeting different KCNJ5 epitopes

• Cross-reactivity assessment:

  • Test against related channels: Check for cross-reactivity with other Kir family members, particularly KCNJ3/GIRK1

  • Species-specific validation: When working with animal models, confirm specificity for the species-specific KCNJ5 ortholog

• Technical controls:

  • Secondary-only controls: Ensure background fluorescence is not contributing to perceived signal

  • Isotype controls: Use rabbit IgG-FITC at matching concentration to assess non-specific binding

This systematic validation approach helps ensure that experimental findings truly reflect KCNJ5 biology rather than antibody artifacts or cross-reactivity.

What are the optimal tissue preparation protocols for KCNJ5 immunodetection in cardiac and adrenal tissues?

Detecting KCNJ5 in cardiac and adrenal tissues requires careful optimization of preparation protocols due to the protein's membrane localization and tissue-specific expression patterns. The following protocol recommendations are based on experimental experience with KCNJ5 Antibody, FITC conjugated:

For Cardiac Tissue:

• Fixation: 4% paraformaldehyde for 24 hours at 4°C (avoid over-fixation which can mask epitopes)

• Cryoprotection: 30% sucrose gradient (10%, 20%, then 30%) before OCT embedding

• Section thickness: 5-8 μm for optimal antibody penetration

• Antigen retrieval: Citrate buffer (pH 6.0) heat-mediated retrieval (95°C for 20 minutes)

• Permeabilization: 0.1% Triton X-100 for 10 minutes at room temperature

• Blocking: 10% normal goat serum, 1% BSA in PBS for 1 hour

• Primary antibody: KCNJ5 Antibody, FITC conjugated at 1:100-1:200 dilution, overnight at 4°C

• Counterstaining: DAPI for nuclei and wheat germ agglutinin for membrane definition

For Adrenal Tissue:

• Fixation: 2% paraformaldehyde for 12 hours at 4°C (reduced fixation preserves zona glomerulosa epitopes)

• Embedding: Paraffin embedding with controlled temperature not exceeding 56°C

• Section thickness: 3-5 μm (thinner sections improve signal-to-noise ratio)

• Deparaffinization: Standard xylene and ethanol series

• Antigen retrieval: Tris-EDTA buffer (pH 9.0) for 30 minutes at 95°C

• Permeabilization: 0.2% Triton X-100 for 15 minutes

• Blocking: 5% BSA with 0.1% Tween-20 for 1 hour

• Primary antibody: KCNJ5 Antibody, FITC conjugated at 1:100 dilution, overnight at 4°C

• Sudan Black B treatment: 0.1% in 70% ethanol for 20 minutes to reduce autofluorescence

These optimized protocols help maximize specific KCNJ5 detection while minimizing background and preserving tissue morphology in the two principal tissues where KCNJ5 has critical physiological functions.

How can researchers troubleshoot weak or absent FITC signal when using KCNJ5 Antibody, FITC conjugated?

When encountering weak or absent FITC signal with KCNJ5 Antibody, FITC conjugated, systematically troubleshoot using this decision tree approach:

• Antibody Integrity Assessment:

  • Check fluorescence of undiluted antibody stock by spotting on filter paper

  • Verify storage conditions (-20°C, protected from light)

  • Test expiration date and freeze-thaw history

• Epitope Accessibility Problems:

  • Optimize fixation: Overfixation can mask epitopes; try reduced fixation times

  • Enhance antigen retrieval: Test different buffers (citrate pH 6.0 vs. Tris-EDTA pH 9.0)

  • Adjust permeabilization: Increase detergent concentration or duration for intracellular epitopes

• Protocol Optimization:

  • Increase antibody concentration: Test serial dilutions (1:50, 1:100, 1:200)

  • Extend incubation time: Try room temperature for 2 hours vs. overnight at 4°C

  • Add signal amplification: Consider tyramide signal amplification (TSA) compatible with FITC

• Technical Considerations:

  • Check microscope settings: Ensure proper excitation (490nm) and emission (525nm) filter sets

  • Adjust exposure settings: Increase exposure time while monitoring background

  • Reduce photobleaching: Add anti-fade reagents and minimize exposure during handling

• Biological Variables:

  • Verify KCNJ5 expression: Confirm expression using RT-PCR in your specific sample

  • Consider protein conformation: The specific epitope (AA 348-419 or 350-450) might be masked in certain conditions

  • Test positive controls: Include samples with known high KCNJ5 expression

By systematically working through these troubleshooting steps, researchers can identify and address the specific factors limiting FITC signal detection in their KCNJ5 immunofluorescence experiments.

What standardization methods ensure reproducible quantification of KCNJ5 expression across different experimental batches?

Achieving reproducible quantification of KCNJ5 expression across experimental batches requires rigorous standardization. Implement these methods to enhance reproducibility:

• Antibody Lot Validation and Normalization:

  • Create a standard curve for each new antibody lot using control samples

  • Maintain a reference sample set that is processed with each experimental batch

  • Document lot numbers and establish correction factors between lots

• Instrument Calibration and Settings Standardization:

  • For fluorescence microscopy: Use calibration beads to normalize intensity values

  • For flow cytometry: Implement daily QC with fluorescent beads to track instrument performance

  • For Western blotting: Include gradient standard curves on each gel

• Data Acquisition Protocols:

  • Standardize image acquisition parameters (exposure time, gain, offset)

  • Establish fixed dynamic range settings across experiments

  • Use automated acquisition where possible to reduce operator variability

• Internal Controls and Normalization:

  • Include housekeeping protein controls appropriate for your experimental system

  • Implement ratiometric analysis (KCNJ5 signal/housekeeping signal)

  • Consider spike-in controls with known quantities of recombinant protein

• Data Analysis Standardization:

  • Use validated analysis pipelines with defined thresholding criteria

  • Implement blind analysis when possible to reduce bias

  • Document all analysis parameters for reproducibility

These standardization approaches collectively minimize technical variability, enabling more reliable detection of true biological differences in KCNJ5 expression across experimental conditions.

What is the optimal methodology for dual immunolabeling studies examining KCNJ5 interactions with regulatory proteins?

Dual immunolabeling studies investigating KCNJ5 interactions with regulatory proteins require careful experimental design to generate reliable co-localization data. The optimal methodology includes:

• Primary Antibody Selection and Validation:

  • Choose antibodies from different host species (e.g., rabbit anti-KCNJ5-FITC with mouse anti-regulatory protein)

  • If using same-species antibodies, implement sequential labeling with blocking steps

  • Validate each antibody individually before dual labeling

• Fluorophore Selection:

  • Pair FITC (excitation: 490nm, emission: 525nm) with spectrally distinct fluorophores

  • Recommended pairs: FITC + Cy3 (low overlap) or FITC + Alexa 647 (minimal overlap)

  • Consider fluorophore brightness matching for balanced signal detection

• Sample Preparation Optimization:

  • Determine compatible fixation for both targets (typically 2-4% PFA works well)

  • Test different permeabilization conditions that maintain epitope accessibility for both targets

  • Optimize blocking to minimize non-specific binding (5% normal serum from both secondary antibody host species)

• Acquisition Protocol:

  • Use sequential scanning on confocal microscopy to eliminate bleed-through

  • Acquire z-stacks with Nyquist sampling for accurate 3D co-localization analysis

  • Include single-labeled controls in each experiment for channel alignment verification

• Quantitative Co-localization Analysis:

  • Calculate multiple co-localization parameters (Pearson's, Mander's coefficients)

  • Implement intensity correlation analysis to distinguish coincidental from biological co-localization

  • Use object-based co-localization for discrete structures

• Functional Validation:

  • Complement imaging with proximity ligation assay for protein-protein interactions within 40nm

  • Validate interactions with co-immunoprecipitation using the same antibodies

  • Consider FRET analysis for direct interaction studies

This comprehensive approach generates robust data on KCNJ5's spatial relationships with regulatory proteins, enabling insights into channel regulation and complex formation in different physiological states.

How can researchers effectively use KCNJ5 Antibody, FITC conjugated in patch-clamp fluorometry experiments?

Patch-clamp fluorometry (PCF) combines electrophysiological recording with fluorescence imaging, providing powerful insights into structure-function relationships of ion channels. For effective use of KCNJ5 Antibody, FITC conjugated in PCF experiments:

• Experimental Setup Optimization:

  • Use an upright microscope with water-immersion objectives for patch-clamp compatibility

  • Configure optical filters for FITC detection (excitation: 490nm, emission: 525nm)

  • Implement high-sensitivity detection (EMCCD or sCMOS camera) for minimal exposure requirements

• Antibody Application Strategies:

  • External epitope labeling: Apply diluted antibody (1:200-1:500) in external solution before patching

  • Internal epitope access: Include diluted antibody (1:500-1:1000) in patch pipette solution

  • For trafficking studies: Pre-label cells, establish recording, then monitor fluorescence redistribution

• Protocol Considerations:

  • Antibody binding confirmation: Allow 10-15 minutes for binding before electrical recording

  • Reduced antibody concentration: Use more dilute solutions than for imaging alone to prevent channel function interference

  • Control recordings: Compare channel properties in labeled vs. unlabeled cells to verify antibody doesn't alter function

• Signal Collection and Analysis:

  • Synchronize electrophysiology and optical data acquisition

  • Implement low-light imaging protocols to minimize photobleaching during extended recordings

  • Correlate fluorescence intensity changes with electrophysiological events

• Controls and Validations:

  • Peptide competition controls: Pre-absorb antibody with immunizing peptide to confirm specificity

  • Fluorescence-only controls: Monitor FITC signal stability without electrical manipulation

  • Electrophysiology-only controls: Verify channel function without fluorophore excitation

This integrated approach allows researchers to correlate KCNJ5 localization, conformational changes, or trafficking with channel function in real-time, providing unique insights into channel regulation that cannot be obtained through either technique alone.