WNK3 Antibody, FITC conjugated

What is WNK3 and what role does it play in cellular physiology?

WNK3 (WNK Lysine Deficient Protein Kinase 3) is a serine/threonine kinase that plays crucial roles in the regulation of electrolyte homeostasis, cell signaling, survival, and proliferation . It belongs to the "with no lysine (K)" family of serine-threonine protein kinases, which lack the catalytic lysine in subdomain II but instead have a conserved lysine in subdomain I .

WNK3 functions as:

• A positive regulator of sodium-coupled chloride cotransporters (NCC, NKCC2)

• An inhibitor of potassium-coupled chloride cotransporters

• A regulator of Ca²⁺ influx by enhancing TRPV5 and TRPV6 membrane expression

• An inhibitor of KCNJ1 by decreasing its cell membrane expression

Unlike other WNK family members (WNK1, WNK4), WNK3 is expressed throughout the nephron, predominantly at intercellular junctions, suggesting its broader role in renal physiology .

What are the key specifications of commercially available FITC-conjugated WNK3 antibodies?

Commercial FITC-conjugated WNK3 antibodies typically feature the following specifications:

For research applications, it's important to note that some products may be custom manufactured with lead times of 3-4 weeks .

What are the optimal storage conditions for maintaining FITC-conjugated WNK3 antibody activity?

To maintain optimal activity of FITC-conjugated WNK3 antibodies, researchers should adhere to these storage recommendations:

• Store at -20°C for long-term storage (typically stable for 12 months from receipt)

• Some products may be stored at 4°C for shorter periods

• Aliquot the antibody upon receipt to avoid repeated freeze-thaw cycles, which can degrade antibody quality

• Protect from continuous light exposure, as this causes gradual loss of fluorescence in FITC conjugates

• Store in the provided buffer containing stabilizers (typically PBS with 50% glycerol and 0.02% sodium azide)

• When handling, keep the antibody on ice and minimize exposure to room temperature

These precautions will help maintain both the antibody binding capacity and the fluorescence properties of the FITC conjugate.

How should researchers design proper controls when using FITC-conjugated WNK3 antibodies?

When designing controls for experiments using FITC-conjugated WNK3 antibodies, researchers should include:

• Negative controls:

  • Secondary antibody-only control to assess non-specific binding

  • Isotype control (rabbit IgG-FITC) to estimate background from non-specific binding

  • Peptide competition assay using the immunizing peptide to confirm specificity

  • Unstained samples to establish autofluorescence baseline

• Positive controls:

  • Tissues or cell lines with known WNK3 expression (kidney tissues for nephron studies)

  • Transfection controls with WNK3 overexpression constructs

  • Previously validated samples with established WNK3 detection patterns

• Technical controls:

  • Titration series to determine optimal antibody concentration (typically 1:500 for immunofluorescence)

  • Single-color controls for spectral compensation when using multiple fluorophores

  • Vehicle controls when studying effects of treatments on WNK3 expression

Implementing these controls will help validate experimental findings and distinguish true WNK3 signal from technical artifacts.

How does WNK3 regulate ion cotransporters, and how can FITC-conjugated antibodies help elucidate these mechanisms?

WNK3 exhibits complex regulatory effects on ion cotransporters that can be mechanistically studied using FITC-conjugated antibodies:

Mechanism of WNK3 regulation:

• Kinase-active WNK3 acts as a potent activator of both NKCC2 and NCC-mediated transport (>3-fold increase in activity)

• Conversely, kinase-inactive WNK3 functions as a potent inhibitor of NKCC2 and NCC activity (≈85% inhibition)

• WNK3 regulates these transporters by altering their expression at the plasma membrane - increasing surface expression in its kinase-active state and decreasing it when catalytically inactive

• WNK3 increases NKCC2 phosphorylation at Thr-184 and Thr-189, sites required for vasopressin-mediated translocation and activation

Methodological approach using FITC-conjugated WNK3 antibodies:

• Co-localization studies: Use FITC-WNK3 antibodies alongside antibodies against ion transporters (using different fluorophores) to visualize spatial relationships

• Response to stimuli: Track WNK3 translocation following vasopressin stimulation or osmotic challenges

• Phosphorylation dynamics: Combine with phospho-specific antibodies to correlate WNK3 activity with transporter phosphorylation status

• Live-cell imaging: Monitor kinetics of WNK3 recruitment to membrane compartments during signaling events

These approaches can provide insights into how WNK3 serves as a molecular switch coordinating diverse ion transport pathways to maintain homeostasis during physiological perturbation .

What methodological considerations are important when optimizing immunofluorescence protocols with FITC-conjugated WNK3 antibodies?

Optimizing immunofluorescence protocols with FITC-conjugated WNK3 antibodies requires attention to several technical considerations:

Sample preparation:

• Fixation method significantly impacts epitope availability (4% paraformaldehyde for 20 minutes is often suitable)

• Permeabilization conditions should be optimized (0.1-0.5% Triton X-100 for 5-10 minutes)

• Blocking solution with 10% fetal bovine serum in PBS for 20 minutes helps reduce non-specific binding

Antibody application:

• Recommended dilution typically ranges from 1:50-1:500 depending on the specific antibody and application

• Incubation should be performed in the dark to prevent photobleaching of FITC

• Optimal incubation time is generally 1 hour at room temperature or overnight at 4°C

Signal enhancement and preservation:

• Anti-fading mounting media is essential for preserving FITC signal during microscopy

• Consider using TSA (tyramide signal amplification) for low-abundance targets

• Minimize exposure to excitation light during imaging to prevent photobleaching

Confocal settings optimization:

• Use appropriate excitation (~495 nm) and emission (~520 nm) filter settings for FITC

• Adjust laser power, detector gain, and pinhole size to minimize background while maintaining signal

• Consider spectral unmixing when using multiple fluorophores to account for FITC's relatively broad emission spectrum

Following these considerations will help maximize signal-to-noise ratio and ensure reliable detection of WNK3 protein in cellular contexts.

How can FITC-conjugated WNK3 antibodies be used to investigate the role of WNK3 in cancer immunotherapy research?

Recent discoveries highlighting WNK3's role in cancer immunotherapy open new research avenues where FITC-conjugated WNK3 antibodies can be valuable tools:

Research context:

• WNK3 functions as a positive regulator of PD-L1 expression, a key immune checkpoint protein

• WNK3 inhibition enhances CD8+ T-cell-mediated antitumor activity and suppresses tumor growth

• The c-JUN N-terminal kinase (JNK)/c-JUN pathway underlies WNK3-mediated transcriptional regulation of PD-L1

Methodological applications of FITC-WNK3 antibodies:

• Tumor microenvironment studies:

  • Multiplex immunofluorescence to visualize WNK3, PD-L1, and T-cell markers simultaneously in tumor tissues

  • FACS analysis of tumor-infiltrating lymphocytes and cancer cells to quantify WNK3 expression across cell populations

• Mechanism investigation:

  • Co-staining with phospho-JNK and phospho-c-JUN antibodies to evaluate activation of this pathway in relation to WNK3 expression

  • Time-course imaging of WNK3 localization during T-cell/cancer cell interactions

• Drug development:

  • High-content screening to identify compounds that modulate WNK3 expression or localization

  • Validation of WNK3 inhibitor (e.g., WNK463) effects on WNK3 expression patterns

• Predictive biomarker development:

  • Analysis of WNK3 expression in patient samples to correlate with immunotherapy response

  • Development of standardized WNK3 detection protocols for potential diagnostic applications

This approach allows researchers to explore WNK3 inhibition as a potential therapeutic strategy for cancer immunotherapy through its concurrent impact on cancer cells and immune cells .

What are the technical challenges when performing Western blot analysis with FITC-conjugated WNK3 antibodies?

Western blot analysis using FITC-conjugated WNK3 antibodies presents unique technical challenges that researchers should address:

Challenges and solutions:

• Signal detection:

  • FITC fluorescence may not be optimal for Western blot detection compared to chemiluminescence

  • Solution: Use a fluorescent scanner with appropriate excitation/emission settings for FITC detection

  • Alternative: Consider using unconjugated primary WNK3 antibody with HRP-conjugated secondary for traditional chemiluminescence detection

• Molecular weight verification:

  • WNK3 is a large protein (~1800 amino acids, ~200 kDa)

  • Solution: Use gradient gels (4-15%) and extended separation times for proper resolution

  • Include appropriate molecular weight markers covering high molecular weight range

• Cross-reactivity concerns:

  • FITC-conjugated antibodies may show non-specific binding

  • Solution: Validate specificity using WNK3 knockout/knockdown samples

  • Include peptide competition controls where the antibody is pre-incubated with the immunizing peptide

• Sample preparation considerations:

  • WNK kinases are sensitive to dephosphorylation during sample preparation

  • Solution: Include phosphatase inhibitors in lysis buffers

  • Maintain cold conditions throughout sample preparation

• Loading control selection:

  • Traditional loading controls may not be appropriate for all experimental conditions

  • Solution: Consider total protein staining methods like Ponceau S alongside specific loading controls

Recommended dilutions for Western blot applications using WNK3 antibodies typically range from 1:500-1:2000 , but should be empirically determined for each specific FITC-conjugated antibody.

How does the kinase activity of WNK3 influence its functions, and what methods can assess this activity?

The kinase activity of WNK3 is central to its function, with distinct outcomes depending on its activation state:

Kinase-dependent functions:

• Kinase-active WNK3 increases NCC and NKCC2 activity (>3-fold), while kinase-inactive WNK3 inhibits them by ~85%

• WNK3 increases Ca²⁺ influx via TRPV5/TRPV6 through a kinase-dependent pathway enhancing membrane expression

• The kinase-dead WNK3 mutant fails to elevate PD-L1 levels, indicating kinase domain involvement in immunoregulation

Methodological approaches to assess kinase activity:

• Autophosphorylation assays:

  • Express WNK3-HA in cells, immunoprecipitate with anti-HA agarose beads

  • Incubate with γ-³²P ATP in kinase buffer at 30°C for 30 minutes

  • Resolve by SDS-PAGE and visualize by autoradiography

• Substrate phosphorylation analysis:

  • Co-express WNK3 with known substrates (e.g., WNK4, SLC12A1, SLC12A2)

  • Assess phosphorylation using phospho-specific antibodies

  • Compare effects of wild-type WNK3 versus kinase-dead mutant (D294A)

• Inhibitor studies:

  • Utilize pan-WNK inhibitors like WNK463 to pharmacologically inhibit WNK3

  • Measure downstream effects on target protein phosphorylation or membrane expression

  • Assess functional outcomes like ion transport or PD-L1 expression

• Cellular localization:

  • Use FITC-conjugated WNK3 antibodies to track localization changes associated with kinase activity

  • Correlate localization with active (phosphorylated) states of the protein

These approaches help elucidate the mechanisms by which WNK3 functions as a molecular switch in cellular signaling and ion transport regulation, with important implications for both physiological processes and pathological conditions.

What are common issues when using FITC-conjugated antibodies and how can researchers overcome them?

When working with FITC-conjugated WNK3 antibodies, researchers may encounter several common issues:

Issue: Rapid photobleaching

• Solution: Minimize exposure to light during all steps of the protocol

• Add anti-fade agents to mounting media

• Use lower excitation intensity during imaging with shorter exposure times and multiple frame averaging

• Store slides in the dark at 4°C

Issue: High background fluorescence

• Solution: Increase blocking time (up to 60 minutes) with 10% FBS or BSA

• Optimize antibody concentration through titration experiments

• Include 0.1% Tween-20 in wash buffers

• Ensure complete washing between steps (at least 3 x 5 minutes)

• Consider using Sudan Black B to reduce autofluorescence in certain tissues

Issue: Weak signal

• Solution: Extend primary antibody incubation time (overnight at 4°C)

• Optimize fixation method (overfixation can mask epitopes)

• Try antigen retrieval methods if applicable

• Consider signal amplification systems for low-abundance targets

Issue: Non-specific binding

• Solution: Use peptide competition controls to confirm specificity

• Include appropriate blocking of endogenous biotin/avidin if using biotin-based detection systems

• Pre-adsorb antibody with tissues/cells lacking the target

• Include isotype controls to establish background levels

Issue: Spectral overlap in multi-color experiments

• Solution: Carefully select fluorophore combinations with minimal spectral overlap

• Perform proper compensation controls

• Consider sequential imaging rather than simultaneous acquisition

• Use spectral unmixing algorithms during image analysis

Addressing these common issues through systematic optimization will help ensure reliable and reproducible results when using FITC-conjugated WNK3 antibodies in research applications.

How can researchers validate the specificity of FITC-conjugated WNK3 antibodies?

Validating antibody specificity is crucial for reliable experimental results. For FITC-conjugated WNK3 antibodies, consider these validation approaches:

Genetic validation:

• Test antibody in WNK3 knockout or knockdown models (siRNA, CRISPR/Cas9)

• Compare staining patterns in wild-type versus WNK3-deficient samples

• Evaluate antibody performance in WNK3-overexpressing systems

Peptide competition:

• Pre-incubate the antibody with excess immunizing peptide (3-fold molar excess)

• Compare staining pattern with and without peptide competition

• Specific signal should be significantly reduced or eliminated

Cross-reactivity assessment:

• Test antibody against related WNK family members (WNK1, WNK2, WNK4)

• Evaluate staining in tissues with known expression patterns of WNK isoforms

• Use multiple antibodies targeting different epitopes of WNK3 to confirm staining patterns

Correlation with other detection methods:

• Compare immunostaining results with mRNA expression (RNA-seq, RT-PCR)

• Verify protein detection by alternative methods (mass spectrometry)

• Correlate with functional assays that reflect WNK3 activity

Independent antibody comparison:

• Compare staining patterns using antibodies from different vendors or those targeting different epitopes

• Consistent patterns across different antibodies increase confidence in specificity

Documentation and reporting:

• Record detailed validation data following best practices for antibody validation

• Include validation controls in experimental reports and publications

• Provide complete antibody information (catalog number, lot, epitope, dilution)

Thorough validation ensures that experimental results truly reflect WNK3 biology rather than artifacts of non-specific binding or cross-reactivity.

How are FITC-conjugated WNK3 antibodies being utilized in cancer immunotherapy research?

Recent research has revealed WNK3 as a novel positive regulator of PD-L1 expression, opening new avenues for cancer immunotherapy research utilizing FITC-conjugated WNK3 antibodies:

Current research applications:

• Mechanistic studies of immune checkpoint regulation:

  • FITC-WNK3 antibodies help visualize how WNK3 regulates the JNK/c-JUN pathway that controls PD-L1 transcription

  • Co-localization studies with PD-L1 and signaling intermediates reveal spatial relationships during immune checkpoint activation

• Tumor microenvironment characterization:

  • Multiparameter flow cytometry with FITC-WNK3 antibodies enables quantification of WNK3 expression across different immune and cancer cell populations

  • Tissue imaging reveals WNK3 distribution patterns in relation to immune infiltrates and checkpoint molecule expression

• Drug development and validation:

  • High-content screening assays incorporating FITC-WNK3 antibodies help identify compounds that modulate WNK3 expression or localization

  • Validation studies for WNK inhibitors like WNK463 assess their effects on WNK3-mediated PD-L1 regulation

• Biomarker development:

  • Standardized protocols using FITC-WNK3 antibodies are being developed to assess WNK3 expression in patient samples

  • Correlation studies examine WNK3 expression patterns in relation to immunotherapy response

Research findings:

• WNK3 perturbation increases cancer cell death in cancer cell-immune cell coculture conditions

• WNK3 inhibition boosts secretion of cytokines and cytolytic enzymes from CD4+ and CD8+ T cells

• WNK463 (pan-WNK inhibitor) enhances CD8+ T-cell-mediated antitumor activity as monotherapy and in combination with low-dose anti-PD-1 antibody

These applications highlight how FITC-conjugated WNK3 antibodies are instrumental in exploring the potential of WNK3 inhibition as a therapeutic strategy for cancer immunotherapy.

What are the considerations when using FITC-conjugated WNK3 antibodies in multiplex immunofluorescence studies?

Multiplex immunofluorescence involving FITC-conjugated WNK3 antibodies requires careful experimental design:

Spectral considerations:

• FITC has excitation/emission maxima around 495/520 nm, which may overlap with other green fluorophores

• Pair FITC with fluorophores that have minimal spectral overlap (e.g., Cy3, Cy5, APC)

• Consider using fluorophores with narrow emission spectra for cleaner separation

• Sequential imaging may be necessary if spectral overlap cannot be resolved through filter selection

Panel design:

• Select markers that answer specific biological questions about WNK3 function

• For ion transport studies: combine with NCC, NKCC2, or other transporters

• For cancer immunotherapy: include PD-L1, T-cell markers, and JNK/c-JUN pathway components

• Include nuclear counterstain (DAPI/Hoechst) that doesn't interfere with FITC signal

Technical optimization:

• Carefully titrate each antibody in the panel individually before combining

• Test for antibody cross-reactivity and species compatibility

• Establish a staining sequence that minimizes interference (typically from lowest to highest abundance targets)

• Include single-color controls for proper spectral unmixing

Signal amplification considerations:

• If WNK3 signal is weak, consider tyramide signal amplification (TSA) for FITC

• Note that TSA requires HRP-conjugated secondary antibodies rather than direct FITC conjugates

• Sequential TSA can be performed for multiple targets using different fluorophores

Analysis approaches:

• Use software capable of spectral unmixing for accurate signal separation

• Consider automated cell segmentation and quantification for objective analysis

• Develop standardized analysis workflows that can be validated across experiments

These considerations will help researchers generate high-quality multiplex data that accurately reflects the biological relationships between WNK3 and other proteins of interest in both physiological and pathological contexts.

What emerging techniques might enhance the utility of FITC-conjugated WNK3 antibodies in research?

Several emerging techniques promise to expand the research applications of FITC-conjugated WNK3 antibodies:

Super-resolution microscopy:

• Techniques like STORM, PALM, and STED can overcome the diffraction limit of conventional microscopy

• May reveal nanoscale organization of WNK3 in relation to ion transporters at cell junctions

• Could elucidate previously undetectable spatial relationships between WNK3 and signaling partners

Proximity labeling approaches:

• Combining FITC-WNK3 antibody detection with BioID or APEX2 proximity labeling

• Would allow identification of proteins that transiently interact with WNK3

• Could reveal new components of WNK3 signaling networks in specific cellular contexts

Live-cell imaging techniques:

• Development of cell-permeable fluorescent WNK3 nanobodies

• Would enable real-time tracking of endogenous WNK3 dynamics

• Could reveal kinetics of WNK3 redistribution during signaling events

Single-cell technologies:

• Integration with single-cell proteomics approaches

• Would allow correlation of WNK3 expression with cellular phenotypes at unprecedented resolution

• Could identify previously unrecognized cell populations with unique WNK3 expression patterns

Spatial transcriptomics:

• Combining FITC-WNK3 immunofluorescence with spatial transcriptomics

• Would correlate WNK3 protein expression with transcriptional landscapes

• Could reveal feedback mechanisms in WNK3-regulated processes

AI-assisted image analysis:

• Machine learning algorithms for automated quantification of WNK3 localization

• Would enable high-throughput analysis of WNK3 distribution patterns

• Could identify subtle phenotypes missed by conventional analysis approaches