PKD1L3 Antibody, FITC conjugated

What is PKD1L3 and what are its primary biological functions?

PKD1L3 (Polycystic Kidney Disease 1 Like 3) is a multi-pass membrane protein that functions as a component of a calcium channel. It serves as a subunit of a nonselective ion channel complex that contributes to sensory functions, particularly in taste perception. This tetrameric channel complex is thought to play a role in mediating sour taste reception in gustatory cells, though its direct contribution to sour taste perception remains somewhat unclear in vivo and may be indirect .

PKD1L3 is expressed at high levels in the liver and testis, though its specific functions in these tissues remain to be fully elucidated . The protein contains several structural domains including a PLAT domain, a GPS domain, and a C-type lectin domain, suggesting complex regulatory and interaction capabilities .

How does the FITC conjugation affect the functionality of PKD1L3 antibodies in research applications?

FITC (fluorescein isothiocyanate) conjugation to PKD1L3 antibodies creates a directly detectable immunoreagent that eliminates the need for secondary antibody incubation steps in immunofluorescence applications. This conjugation provides researchers with several methodological advantages: direct visualization of the target protein, reduction in non-specific binding that can occur with secondary antibodies, and compatibility with multi-labeling experiments using antibodies from the same host species .

The FITC-conjugated PKD1L3 antibodies maintain their binding specificity to the target epitope (typically AA 121-220 in commercially available options) while gaining fluorescent properties with excitation around 495 nm and emission at approximately 519 nm . It is important to note that researchers should protect these conjugated antibodies from prolonged light exposure during storage and experimental procedures to prevent photobleaching of the fluorophore.

What characteristics distinguish polyclonal PKD1L3 antibodies from other antibody types?

The polyclonal PKD1L3 antibodies available for research are typically raised in rabbits using KLH-conjugated synthetic peptides derived from human PKD1L3 as immunogens . These polyclonal reagents offer several distinct characteristics relevant to research applications:

• Epitope recognition: Polyclonal antibodies recognize multiple epitopes on the PKD1L3 protein, potentially providing stronger signal amplification compared to monoclonal antibodies, especially in tissues where protein expression is low.

• Production methodology: They are purified by Protein A affinity chromatography, yielding IgG fractions with reactivity specific to human PKD1L3 .

• Cross-reactivity profile: Most commercially available polyclonal PKD1L3 antibodies show predicted reactivity with human samples, though cross-reactivity with other species would require experimental validation .

• Application versatility: These antibodies demonstrate utility across various applications including immunofluorescence in cultured cells, paraffin-embedded sections, ELISA, and dot blot techniques .

What are the optimal protocols for using FITC-conjugated PKD1L3 antibodies in immunofluorescence applications?

For successful immunofluorescence applications with FITC-conjugated PKD1L3 antibodies, researchers should follow these methodological guidelines:

For cultured cells (IF (cc)):

• Culture cells on appropriate cover slips or chamber slides until 70-80% confluence

• Fix cells with 4% paraformaldehyde (15 minutes at room temperature)

• Permeabilize with 0.1-0.5% Triton X-100 (10 minutes)

• Block with 5% normal serum or BSA (1 hour)

• Incubate with FITC-conjugated PKD1L3 antibody at 1:50-1:200 dilution (overnight at 4°C or 1-2 hours at room temperature)

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

For paraffin-embedded tissue sections (IF (p)):

• Deparaffinize and rehydrate tissue sections

• Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

• Block endogenous peroxidase activity with 3% H₂O₂

• Block with 10% normal serum

• Incubate with FITC-conjugated PKD1L3 antibody at 1:50-1:100 dilution (overnight at 4°C)

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

The visualization should be conducted using a fluorescence microscope with appropriate filter sets for FITC detection (excitation: ~495 nm, emission: ~519 nm).

What controls should be incorporated when using FITC-conjugated PKD1L3 antibodies?

When conducting experiments with FITC-conjugated PKD1L3 antibodies, the following controls are essential for result validation and experimental rigor:

Positive Controls:

• Human placenta, heart, or lung tissues where PKD1L3 expression has been documented

• A549 (human lung carcinoma) cell line, which has been confirmed to express PKD1L3

Negative Controls:

• Omission of primary antibody to assess background autofluorescence

• Isotype control (FITC-conjugated rabbit IgG) to evaluate non-specific binding

• Pre-absorption control using the immunizing peptide to confirm antibody specificity

• Tissues known to lack PKD1L3 expression

Technical Controls:

• DAPI or other nuclear counterstain to verify tissue morphology and cell health

• Positive staining control using a well-characterized antibody against a housekeeping protein

• Concentration gradient tests (antibody titration) to determine optimal working dilution

How can PKD1L3 antibodies be integrated into multi-labeling immunofluorescence protocols?

For multi-labeling studies incorporating FITC-conjugated PKD1L3 antibodies, consider the following methodological approach:

Sequential Protocol for Multiple Antibodies:

• Planning phase:

  • Select complementary fluorophores with minimal spectral overlap (e.g., FITC for PKD1L3, Cy3 or TRITC for second target, Cy5 or APC for third target)

  • Choose antibodies raised in different host species when using unconjugated primary antibodies

  • Plan for nuclear counterstain (DAPI or Hoechst) compatible with your fluorophore selection

• Staining procedure:

  • Perform fixation and antigen retrieval as described above

  • Block with serum from all secondary antibody host species

  • Option 1 (simultaneous incubation): Apply FITC-conjugated PKD1L3 antibody together with other primary antibodies if raised in different species

  • Option 2 (sequential incubation): Apply antibodies sequentially with washing steps in between if cross-reactivity is a concern

  • Add appropriate secondary antibodies for unconjugated primaries

  • Apply nuclear counterstain

  • Mount with anti-fade medium

• Analysis considerations:

  • Use single-labeled controls to set up proper compensation when using fluorophores with overlapping spectra

  • Capture images sequentially rather than simultaneously to minimize bleed-through

  • Consider spectral unmixing during image analysis if using fluorophores with similar emission spectra

What are common causes of high background or non-specific signal when using FITC-conjugated PKD1L3 antibodies?

When using FITC-conjugated PKD1L3 antibodies, several factors can contribute to elevated background or non-specific signals:

• Fixation issues:

  • Over-fixation can increase tissue autofluorescence, particularly with aldehyde-based fixatives

  • Under-fixation may result in poor morphology and antigen preservation

• Antibody concentration:

  • Excessive antibody concentration leads to non-specific binding and high background

  • Insufficient dilution of the FITC-conjugated PKD1L3 antibody (recommended working dilutions typically range from 1:50 to 1:200)

• Blocking inadequacies:

  • Insufficient blocking of non-specific binding sites

  • Inappropriate blocking agent for the tissue type

• Technical factors:

  • Tissue drying during incubation steps

  • Insufficient washing between steps

  • Contamination of buffers or reagents

  • Photobleaching due to excessive light exposure

• Tissue-specific concerns:

  • Natural autofluorescence in certain tissues (particularly liver, kidney, and brain)

  • High lipofuscin content in older tissues causing autofluorescence in the FITC channel

What optimization strategies can improve signal-to-noise ratio in PKD1L3 immunofluorescence experiments?

To enhance signal-to-noise ratio when working with FITC-conjugated PKD1L3 antibodies:

Antibody Optimization:

• Perform antibody titration experiments using a dilution series (e.g., 1:25, 1:50, 1:100, 1:200, 1:400) to determine optimal concentration

• Extend incubation time at 4°C (overnight) rather than higher temperatures for more specific binding

Protocol Enhancements:

• Increase blocking stringency:

  • Extend blocking time to 1-2 hours

  • Use 5-10% normal serum with 1% BSA and 0.1-0.3% Triton X-100

  • Add 0.1% Tween-20 to washing buffers

• Reduce autofluorescence:

  • Treat sections with 0.1-1% sodium borohydride for 10 minutes before blocking

  • Incubate with 0.1-0.3% Sudan Black B in 70% ethanol after antibody incubation

  • Consider using commercial autofluorescence quenching reagents

• Improve signal detection:

  • Use mounting media specifically designed to preserve fluorescence

  • Optimize image acquisition settings (exposure time, gain, offset)

  • Consider using spectral imaging and linear unmixing to separate specific signal from autofluorescence

How should researchers validate the specificity of PKD1L3 antibody in their experimental system?

To ensure the specificity of PKD1L3 antibody staining, researchers should implement a multi-faceted validation strategy:

Experimental Validation Methods:

• Peptide competition/neutralization assay:

  • Pre-incubate the PKD1L3 antibody with excess immunizing peptide

  • Apply to adjacent tissue sections

  • Specific staining should be abolished or significantly reduced

• Genetic approaches:

  • Use PKD1L3 knockdown/knockout models as negative controls

  • Compare staining in tissues with known differential expression

  • Employ RNAi or CRISPR techniques to reduce PKD1L3 expression and confirm corresponding reduction in antibody signal

• Orthogonal detection methods:

  • Correlate protein detection with mRNA expression (qRT-PCR or in situ hybridization)

  • Confirm localization with different antibodies targeting distinct epitopes of PKD1L3

  • Validate with non-antibody detection methods (e.g., fluorescent protein tagging)

• Molecular weight verification:

  • Perform western blot using the same antibody to confirm detection of a protein band at the expected molecular weight (~200 kDa for full-length PKD1L3)

How can PKD1L3 antibodies contribute to investigating its role in acid sensing and taste perception?

PKD1L3 antibodies offer valuable tools for investigating the protein's proposed role in sour taste perception and acid sensing through several advanced research approaches:

Tissue-Specific Localization Studies:

• Use FITC-conjugated PKD1L3 antibodies to precisely map the expression pattern in taste buds, focusing on specific papillae types (fungiform, foliate, circumvallate)

• Employ co-localization studies with markers for different taste cell types to determine which specific cell populations express PKD1L3

• Compare expression patterns in different species to understand evolutionary conservation of acid sensing mechanisms

Functional Analysis:

• Combine immunofluorescence with calcium imaging in taste cells to correlate PKD1L3 expression with acid-induced calcium responses

• Examine changes in PKD1L3 localization or expression following acid stimulation to assess dynamic regulation

• Investigate potential interactions with PKD2L1, its proposed channel partner, through proximity ligation assays or co-immunoprecipitation followed by immunofluorescence

Structural Investigation:

• Use super-resolution microscopy with PKD1L3 antibodies to examine the spatial organization of PKD1L3 in relation to other channel components

• Analyze the distribution of PKD1L3 in relation to synaptic connections between taste cells and gustatory nerve fibers

• Study the truncated PKD1L3/PKD2L1 complex that retains both Ca²⁺ and acid-induced channel activities

What techniques can be combined with PKD1L3 immunofluorescence to study its calcium channel functionality?

Researchers can employ several complementary techniques alongside FITC-conjugated PKD1L3 antibody staining to investigate its calcium channel functionality:

Functional Calcium Imaging:

• Combine PKD1L3 immunofluorescence with calcium indicators (Fluo-4, Fura-2, or genetically encoded calcium indicators like GCaMP)

• Perform live cell calcium imaging followed by fixation and PKD1L3 immunostaining to correlate functional responses with protein expression

• Use ratiometric calcium imaging to quantify calcium flux in cells expressing PKD1L3

Electrophysiological Approaches:

• Patch-clamp recordings in cells identified by PKD1L3 immunostaining

• Correlate electrophysiological properties with PKD1L3 expression levels quantified by immunofluorescence

• Assess ion selectivity in cells with confirmed PKD1L3 expression

Molecular Structure-Function Analysis:

• Use domain-specific antibodies to different regions of PKD1L3 to understand structure-function relationships

• Combine with site-directed mutagenesis of key amino acids followed by immunolocalization to assess trafficking and channel assembly

• Investigate the interaction between PKD1L3 and PKD2L1 in the formation of functional calcium channels

Experimental Data Table: Comparing Methods for PKD1L3 Functional Analysis

What research approaches can elucidate PKD1L3's functions in liver and testis where it's highly expressed?

To investigate the functions of PKD1L3 in liver and testis tissues where it shows high expression levels , researchers can implement these methodological approaches:

Cellular Characterization:

• Use FITC-conjugated PKD1L3 antibodies to identify specific cell types expressing the protein within these complex tissues

• Perform dual immunofluorescence with cell-type-specific markers:

  • For liver: hepatocytes (HNF4α), cholangiocytes (CK19), Kupffer cells (CD68), stellate cells (GFAP)

  • For testis: Sertoli cells (SOX9), Leydig cells (3β-HSD), spermatogonia (PLZF), spermatocytes (SYCP3)

• Quantify expression levels across developmental stages and in response to physiological stimuli

Functional Analysis:

• Correlate PKD1L3 expression with calcium signaling in isolated primary cells from these tissues

• Assess changes in PKD1L3 localization during physiological processes (e.g., bile secretion in liver, spermatogenesis stages in testis)

• Investigate phenotypic effects of PKD1L3 knockdown/knockout in these tissues, followed by immunofluorescence analysis of potential compensatory mechanisms

Disease-Related Studies:

• Compare PKD1L3 expression patterns between normal and diseased tissues (e.g., fatty liver disease, testicular cancer) using the FITC-conjugated antibodies

• Assess co-localization with disease markers to establish potential pathophysiological roles

• Correlate changes in PKD1L3 expression with functional outcomes in disease models

What are the relative advantages of immunofluorescence versus other techniques for PKD1L3 detection?

When considering methodological approaches for PKD1L3 detection, researchers should evaluate the comparative advantages of immunofluorescence using FITC-conjugated antibodies against alternative techniques:

Comparative Analysis of PKD1L3 Detection Methods:

FITC-conjugated PKD1L3 antibodies excel in applications requiring spatial information about protein distribution while preserving tissue architecture. They are particularly valuable for analyzing heterogeneous tissues like taste buds, liver, and testis where PKD1L3 may have cell type-specific functions .

How should researchers integrate PKD1L3 protein detection with transcriptomic and functional studies?

A comprehensive research approach to PKD1L3 should integrate protein detection with transcriptomic and functional analyses through these methodological strategies:

Multi-omics Integration Framework:

• Correlation of protein and mRNA expression:

  • Perform PKD1L3 immunofluorescence and RNA-seq/qRT-PCR on adjacent tissue sections

  • Create tissue maps correlating protein localization with mRNA expression levels

  • Investigate potential post-transcriptional regulation by comparing protein/mRNA ratios

• Structure-function relationships:

  • Use domain-specific antibodies to different regions of PKD1L3

  • Correlate localization patterns with functional outcomes in calcium imaging or electrophysiology

  • Investigate how structural features of PKD1L3 impact its interaction with PKD2L1 and formation of functional channels

• Systems biology approach:

  • Combine PKD1L3 immunostaining with phospho-protein detection to map signaling networks

  • Correlate PKD1L3 expression with functional readouts (e.g., calcium transients, membrane potential)

  • Use computational modeling to integrate protein expression data with functional parameters

• Single-cell analysis:

  • Implement single-cell RNA-seq followed by PKD1L3 immunostaining on the same tissue

  • Use spatial transcriptomics approaches combined with PKD1L3 protein detection

  • Correlate single-cell gene expression profiles with PKD1L3 protein levels

What emerging research directions could benefit from FITC-conjugated PKD1L3 antibodies?

Several emerging research areas could benefit from the application of FITC-conjugated PKD1L3 antibodies:

Novel Research Applications:

• PKD1L3 in extrasensory tissues:

  • Investigate the unexplored functions of PKD1L3 in liver and testis where it shows high expression

  • Explore potential roles in other tissues where calcium signaling is crucial for function

  • Assess whether PKD1L3 has tissue-specific interacting partners beyond PKD2L1

• Development and differentiation:

  • Track PKD1L3 expression during embryonic development of sensory systems

  • Study its role in taste cell differentiation and renewal

  • Investigate potential developmental functions in liver and testis organogenesis

• Pathophysiological implications:

  • Examine PKD1L3 expression changes in taste disorders

  • Investigate potential roles in liver pathologies where calcium signaling is disrupted

  • Explore possible functions in testicular development and reproductive disorders

• Therapeutic targeting:

  • Use PKD1L3 antibodies to validate potential drug targets in taste modulation

  • Develop screening assays for compounds affecting PKD1L3/PKD2L1 channel function

  • Explore PKD1L3 as a potential biomarker in specific physiological or pathological conditions

• Structural biology applications:

  • Use antibodies as tools to stabilize protein conformations for structural studies

  • Investigate the architecture of the PKD1L3/PKD2L1 heteromeric complex

  • Study the truncated PKD1L3/PKD2L1 complex that retains both Ca²⁺ and acid-induced channel activities