WNK4 Antibody, HRP conjugated

What is WNK4 and what physiological roles does it play?

WNK4 (WNK lysine deficient protein kinase 4) is a serine/threonine kinase that functions as a critical regulator of electrolyte homeostasis, cell signaling, survival, and proliferation. It primarily regulates the activity of the thiazide-sensitive Na-Cl cotransporter (SLC12A3) through phosphorylation, which appears to prevent membrane trafficking of SLC12A3 . Additionally, WNK4 inhibits the renal K+ channel (KCNJ1) via a kinase-independent mechanism by inducing clathrin-dependent endocytosis that clears the protein from the cell surface . WNK4 essentially acts as a molecular switch that balances NaCl reabsorption and K+ secretion to maintain integrated homeostasis, making it a central player in blood pressure regulation and kidney function .

How does the subcellular localization of WNK4 influence its function?

WNK4 demonstrates tissue-specific and subcellular compartment-specific localization patterns that directly relate to its diverse functions. In kidney tubules, WNK4 expression is confined to the distal convoluted tubule and collecting duct of the distal nephron . Specifically, in the distal convoluted tubule, WNK4 is largely associated with tight junctions, whereas in the collecting duct, WNK4 localizes to both the cytoplasm and tight junctions . In non-renal tissues, WNK4 shows selective expression in polarized epithelia rather than being ubiquitously expressed in all cell types. This distinct localization pattern suggests that WNK4's position at intercellular junctions may facilitate its role in regulating paracellular ion flux across epithelia, while its cytoplasmic presence enables direct interactions with ion transporters and channels .

What is the difference between WNK4 regulation of SLC12A3 versus KCNJ1?

WNK4 utilizes distinct mechanisms to regulate different ion transport proteins. For the thiazide-sensitive Na-Cl cotransporter (SLC12A3), WNK4 exerts its inhibitory effect through a kinase-dependent phosphorylation mechanism that prevents membrane trafficking of the transporter . In contrast, WNK4 inhibits the renal K+ channel KCNJ1 via a kinase-independent mechanism by inducing clathrin-dependent endocytosis . This dual regulatory capability enables WNK4 to independently control sodium reabsorption and potassium secretion, allowing for fine-tuned electrolyte balance in response to varying physiological demands. The contrasting mechanisms highlight the complexity of WNK4's regulatory functions and explain why mutations can lead to distinct phenotypic consequences .

What are the key differences between HRP-conjugated and unconjugated WNK4 antibodies?

HRP-conjugated WNK4 antibodies have horseradish peroxidase directly attached to the antibody molecule, eliminating the need for secondary antibody incubation in applications like Western blotting and ELISA . This conjugation offers several advantages: it reduces protocol time by eliminating the secondary antibody step, minimizes background by reducing non-specific binding that can occur with secondary antibodies, and enables more direct quantification in ELISA applications. Unconjugated WNK4 antibodies require a secondary antibody step but offer greater flexibility for different detection methods and can be used across more applications, including immunofluorescence where HRP conjugation would not be suitable . The choice between these formats should be guided by specific experimental requirements, with HRP-conjugated antibodies being particularly valuable for high-throughput applications requiring rapid detection.

What applications are HRP-conjugated WNK4 antibodies validated for?

HRP-conjugated WNK4 antibodies have been primarily validated for Western blotting (WB) and ELISA applications . For Western blotting, the recommended dilution range is typically 1:300-5000, while ELISA applications require a dilution range of 1:500-1000 . The HRP conjugation provides a direct detection method without requiring secondary antibodies, which is particularly advantageous for these applications. In contrast, unconjugated WNK4 antibodies have broader application ranges including Western blotting, immunohistochemistry (IHC), and immunofluorescence (IF), with each application requiring specific optimization of antibody dilutions to achieve optimal results .

What species reactivity has been confirmed for WNK4 antibodies?

Various WNK4 antibodies show different species reactivity profiles. The HRP-conjugated WNK4 polyclonal antibody (bs-0429R-HRP) has confirmed reactivity with mouse samples and predicted reactivity with human and rat samples . Other WNK4 antibodies, such as 22326-1-AP, have been tested and confirmed to react with human, mouse, and rat samples . This cross-species reactivity is valuable for comparative studies across different model organisms. When selecting a WNK4 antibody for your research, it's essential to verify the validated species reactivity to ensure compatibility with your experimental system and to perform appropriate validation if working with species not previously tested .

How should I optimize Western blot protocols when using WNK4 antibody, HRP conjugated?

For optimal Western blot results with HRP-conjugated WNK4 antibody, several protocol optimizations are crucial. Start with proper sample preparation, ensuring complete protein extraction from tissues containing WNK4 (primarily kidney and polarized epithelia) . Use fresh tissue samples or properly stored protein lysates to prevent degradation. Since WNK4 is a high molecular weight protein (observed at approximately 180 kDa) , use low percentage (7-8%) or gradient (4-15%) polyacrylamide gels to ensure proper separation . For transfer, extend the transfer time or reduce voltage to ensure complete transfer of high molecular weight proteins. When blocking, use 5% non-fat dry milk or BSA in TBST for 1-2 hours at room temperature. For antibody incubation, dilute the HRP-conjugated WNK4 antibody within the recommended range (1:300-5000) , optimizing through titration experiments for your specific samples. Include proper controls, including positive control tissues known to express WNK4 and negative controls. Finally, for detection, use enhanced chemiluminescence (ECL) detection systems with exposure times optimized for your specific signal strength .

What controls should I include when using WNK4 antibody in my experiments?

Rigorous experimental design requires several types of controls when using WNK4 antibodies. First, include positive control samples known to express WNK4, such as kidney tissue (particularly distal convoluted tubule and collecting duct) or cell lines with confirmed WNK4 expression (A375, K-562, or PC-12 cells) . Second, incorporate negative control samples where WNK4 is not expressed or is knocked down. Third, perform technical controls: for Western blots, include a loading control (β-actin, GAPDH) to normalize protein loading; for immunostaining, include secondary-only controls to assess non-specific binding. For advanced validation, consider peptide competition assays where pre-incubation of the antibody with the immunizing peptide should abolish specific signals. When studying WNK4's functional effects, compare results with kinase-dead WNK4 (e.g., WNK4-D318A) to distinguish between kinase-dependent and kinase-independent mechanisms . These comprehensive controls ensure the reliability and specificity of your WNK4 antibody-based findings.

How can I determine the optimal dilution of WNK4 antibody, HRP conjugated for my specific application?

Determining the optimal dilution for HRP-conjugated WNK4 antibody requires systematic titration experiments tailored to your specific samples and detection system. For Western blotting, start with a dilution series within the recommended range (1:300-5000) using identical samples across multiple blots or by cutting a single blot into strips. For ELISA applications, prepare a similar dilution series within the 1:500-1000 range . In both cases, maintain all other experimental conditions constant while varying only the antibody concentration. Evaluate results based on signal-to-noise ratio rather than absolute signal intensity—the optimal dilution should provide clear specific bands or signals with minimal background. For tissues or cell types not previously tested with this antibody, start at the more concentrated end of the recommended range and adjust based on results. Document optimization results carefully for future reference, noting that optimal dilutions may vary between sample types, protein extraction methods, and detection systems.

How can I use WNK4 antibody to study the relationship between WNK4 and ion transporters?

Investigating WNK4's regulation of ion transporters requires sophisticated experimental approaches with WNK4 antibodies. Co-immunoprecipitation experiments can identify direct physical interactions between WNK4 and transporters like SLC12A3 (NCC), KCNJ1 (ROMK), or NKCC1 . For these experiments, use WNK4 antibody to pull down the protein complex and probe with antibodies against potential interacting partners. Proximity ligation assays offer an alternative approach to visualize protein-protein interactions in situ. To study functional relationships, combine WNK4 detection with surface biotinylation assays to quantify how WNK4 affects transporter surface expression . Research has shown that WNK4 inhibits NKCC1 by dramatically reducing its surface expression while not affecting total cellular levels, which can be demonstrated through surface biotinylation combined with Western blotting using WNK4 and transporter-specific antibodies . For mechanistic insights, compare the effects of wild-type WNK4 with those of PHAII-causing mutant WNK4 (e.g., Q562E) or kinase-dead WNK4 (D318A) on transporter activity and localization .

What approaches can I use to study WNK4 phosphorylation states and kinase activity?

Investigating WNK4 phosphorylation states and kinase activity requires specialized techniques beyond basic antibody applications. For detecting WNK4 phosphorylation, use phospho-specific antibodies targeting known regulatory sites, combining with immunoprecipitation to enrich for WNK4 before detection. Alternatively, use Phos-tag™ SDS-PAGE, which can separate phosphorylated from non-phosphorylated proteins without specific phospho-antibodies. To assess WNK4 kinase activity, perform in vitro kinase assays using immunoprecipitated WNK4 and known substrates, measuring substrate phosphorylation through radioisotope incorporation or phospho-specific antibodies. To distinguish between kinase-dependent and kinase-independent functions, compare the effects of wild-type WNK4 with kinase-dead WNK4-D318A on target proteins . For instance, research has shown that WNK4's inhibition of ENaC is maintained with kinase-dead WNK4, demonstrating kinase-independent regulation . Cellular studies can be enhanced by using phosphatase inhibitors during sample preparation to preserve phosphorylation states for accurate analysis.

How can I investigate the role of WNK4 in pseudohypoaldosteronism type II (PHAII) using specific antibodies?

Investigating WNK4's role in PHAII requires a multifaceted approach using WNK4 antibodies in various experimental contexts. Compare the subcellular localization of wild-type versus PHAII-mutant WNK4 (e.g., Q562E) using immunofluorescence or immunohistochemistry to identify potential mislocalization . Functional studies should examine how PHAII mutations affect WNK4's interaction with and regulation of key ion transporters. Research has demonstrated that while wild-type WNK4 inhibits ENaC activity by more than 50%, PHAII-WNK4 (Q562E) shows no significant inhibitory effect . This functional difference can be investigated using electrophysiological measurements in expression systems coupled with Western blotting to confirm equivalent protein expression levels. For in vivo relevance, use WNK4 antibodies in immunohistochemistry of kidney sections from PHAII animal models to assess expression patterns and co-localization with regulated transporters. Biochemical approaches should include co-immunoprecipitation to determine how PHAII mutations alter WNK4's protein-protein interaction network, potentially identifying novel therapeutic targets for this disorder.

Why might I see no signal or weak signal when using WNK4 antibody, HRP conjugated?

Absent or weak signals when using HRP-conjugated WNK4 antibody can stem from multiple sources. First, consider sample-related issues: WNK4 has tissue-specific expression primarily in kidney distal convoluted tubule, collecting duct, and select polarized epithelia , so confirm your samples contain WNK4-expressing tissues. Protein degradation may occur if samples weren't properly preserved with protease inhibitors or kept at appropriate temperatures. Technical issues might include insufficient protein loading, incomplete transfer of high-molecular-weight WNK4 (~180 kDa) , or excessive washing that removes antibody. Antibody-related problems could involve decreased activity due to repeated freeze-thaw cycles—the manufacturer recommends storing at -20°C and aliquoting to avoid freeze-thaw cycles . Detection system failures include expired ECL reagents or improper developer settings. To systematically troubleshoot, include positive control samples known to express WNK4 (kidney tissue, A375, K-562, or PC-12 cells) , optimize protein extraction methods, try increased antibody concentration within recommended ranges (1:300-1:5000 for WB) , and extend exposure times during detection.

What could explain the discrepancy between predicted and observed molecular weight for WNK4?

Discrepancies between WNK4's calculated molecular weight (135 kDa) and observed molecular weight (approximately 180 kDa) can be explained by several factors. Post-translational modifications, particularly phosphorylation, significantly contribute to this difference. WNK4 contains multiple phosphorylation sites that, when modified, can substantially alter protein migration during SDS-PAGE. Additionally, the high proline content in WNK4 can cause anomalous migration patterns in gel electrophoresis, as proline-rich regions often fail to bind SDS efficiently, resulting in reduced electrophoretic mobility. Alternative splicing of WNK4 may also generate isoforms with different molecular weights than predicted from the canonical sequence. To confirm that observed bands truly represent WNK4, validate using multiple approaches: compare patterns across different tissues known to express WNK4, use different antibodies targeting distinct epitopes of WNK4, and consider peptide competition assays. For definitive identification of high-molecular-weight bands, mass spectrometry analysis following immunoprecipitation can confirm protein identity despite migration anomalies.

How can I reduce background when using WNK4 antibody in immunohistochemistry or immunofluorescence?

High background when using WNK4 antibodies in immunostaining can be systematically addressed through protocol optimization. Begin by improving blocking—extend blocking time to 2 hours or overnight using 5-10% normal serum from the same species as the secondary antibody, and add 0.1-0.3% Triton X-100 for better penetration . For antibody incubation, use more dilute antibody solutions within the recommended range (1:20-1:200 for IHC, 1:50-1:200 for IF) and extend incubation time at 4°C to improve specific binding while reducing non-specific interactions. Incorporate additional washing steps with PBS/TBS containing 0.1% Tween-20, using at least 3 washes of 10 minutes each between each step. Consider tissue-specific optimizations: for kidney sections, try antigen retrieval with TE buffer pH 9.0 as an alternative to citrate buffer pH 6.0 . For particularly problematic samples, pre-adsorb the antibody with tissue powder from a species different from your target to remove cross-reactive antibodies. Finally, reduce autofluorescence by treating sections with Sudan Black B (0.1% in 70% ethanol) for 20 minutes or use commercial autofluorescence quenching reagents before mounting when performing immunofluorescence.

How do different fixation methods affect WNK4 detection in immunohistochemistry?

The choice of fixation method significantly impacts WNK4 detection in immunohistochemistry and requires careful optimization. Paraformaldehyde (PFA) fixation (4%) preserves tissue morphology but may mask epitopes through protein cross-linking, necessitating antigen retrieval. For WNK4 specifically, research indicates that antigen retrieval with TE buffer pH 9.0 may provide better results than citrate buffer pH 6.0 . Methanol fixation avoids cross-linking but can denature some proteins, potentially affecting conformation-dependent epitopes. Fresh-frozen sections processed with acetone fixation often provide superior epitope preservation but with compromised morphology. When optimizing fixation for WNK4 detection, perform parallel experiments with each fixation method using identical tissues and antibody dilutions. For kidney tissues specifically, where WNK4 shows discrete localization patterns in distal convoluted tubule and collecting duct , fixation optimization is particularly critical for preserving subcellular localization at tight junctions. Document fixation conditions meticulously since different epitopes (and therefore different WNK4 antibodies) may perform optimally under different fixation conditions.

What approaches can I use to simultaneously detect WNK4 and its interaction partners?

Multi-protein detection involving WNK4 and its interaction partners requires sophisticated co-detection strategies. For fluorescence co-localization, use primary antibodies from different host species (e.g., rabbit anti-WNK4 with mouse anti-SLC12A3), followed by species-specific secondary antibodies conjugated to spectrally distinct fluorophores. Include stringent controls for antibody cross-reactivity and fluorophore bleed-through. For protein-protein interaction analysis, proximity ligation assay (PLA) offers superior sensitivity by generating fluorescent signals only when target proteins are within 40nm of each other, ideal for detecting WNK4 interactions with ion transporters like SLC12A3 or KCNJ1 . Co-immunoprecipitation followed by Western blotting remains the gold standard for biochemical interaction verification—immunoprecipitate with anti-WNK4 antibody and probe blots for suspected interaction partners, or vice versa. For dynamic interactions in living cells, consider fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) using tagged proteins. When studying WNK4's role in protein trafficking, combine surface biotinylation assays with WNK4 co-expression to quantitatively assess how WNK4 affects surface expression of transporters like NKCC1 .

How can I use WNK4 antibodies in developing therapeutic approaches for hypertension?

WNK4 antibodies serve as valuable tools in developing novel therapeutic approaches for hypertension through multiple research applications. For target validation, use WNK4 antibodies in immunohistochemistry and Western blotting to compare WNK4 expression and localization patterns between normotensive and hypertensive models, establishing correlation with disease state. In drug discovery applications, develop high-throughput screening assays using WNK4 antibodies to identify compounds that modulate WNK4 expression, localization, or kinase activity. For mechanistic studies, investigate how potential therapeutic compounds affect WNK4's regulation of key ion transporters like SLC12A3, KCNJ1, and ENaC . Employ surface biotinylation assays combined with WNK4 antibody detection to quantify how candidate drugs modify WNK4's effect on transporter trafficking. When evaluating WNK4-targeting compounds in animal models, use WNK4 antibodies to confirm target engagement and monitor on-target effects in kidney tissues. Since WNK4 mutations cause pseudohypoaldosteronism type II (PHAII) with hypertension , comparing how therapeutics differentially affect wild-type versus mutant WNK4 may provide insights into personalized treatment approaches for different hypertension subtypes.