PKD1L1 Antibody

What cellular structures can be visualized using PKD1L1 antibodies?

PKD1L1 antibodies primarily detect the protein in ciliary structures, as PKD1L1 localizes to the cilium where it interacts with PKD2. When conducting immunofluorescence studies, researchers should expect to observe ciliary staining in node cells during embryonic development . The protein comprises 11 transmembrane domains, a C-terminal intracellular coiled coil, and an N-terminal extracellular region containing REJ and PKD domains . Depending on the epitope targeted by your antibody (particularly N-terminal antibodies), you may visualize different cellular compartments where PKD1L1 fragments may be processed or trafficked.

Which tissues show highest PKD1L1 expression for antibody-based detection?

PKD1L1 expression is strongly detected in the bile duct epithelium of liver tissue . When planning immunohistochemistry experiments, prioritize embryonic node tissue for developmental studies, as well as hepatobiliary structures including intrahepatic bile ducts and gallbladder epithelium. In zebrafish models, PKD1L1 is expressed during early development in Kupffer's vesicle (KV), the laterality-defining organ . Human studies have shown PKD1L1 is strongly expressed in the bile duct epithelium in normal infant liver tissue, while expression appears weak or absent in liver tissue from patients with biliary atresia splenic malformation (BASM) .

How should PKD1L1 antibodies be validated before experimental use?

Validation of PKD1L1 antibodies should include multiple complementary approaches:

• Western blot analysis comparing wild-type tissues with PKD1L1 knockout/mutant tissues

• Immunostaining of cells/tissues with known PKD1L1 expression patterns

• Peptide competition assays to confirm specificity of binding

• Comparison with genetic tagging methods (e.g., GFP-tagged PKD1L1)

For zebrafish studies, validate antibodies using the pkd1l1 hsc117 mutant line with the 4-base-pair deletion in exon 3 . For mouse studies, compare with Pkd1l1 rks mutants, which contain an A-to-G transition at nucleotide 1232 resulting in D411G substitution .

How can PKD1L1 antibodies be used to study protein-protein interactions with PKD2?

PKD1L1 has been demonstrated to physically interact with PKD2, forming a complex important for left-right patterning . For investigating this interaction:

• Use co-immunoprecipitation with PKD1L1 antibodies followed by PKD2 detection

• Perform proximity ligation assays in intact cells using antibodies against both proteins

• Consider the coiled-coil domain of PKD1L1 (amino acids 2440-2607) as a critical region for PKD2 interaction

When designing experiments, note that in mouse studies, systematic comparison of Pkd1l1 and Pkd2 mutants revealed strong phenocopying, supporting their functional interaction . Use antibodies targeting the C-terminal coiled-coil region for interaction studies, as this domain has been shown to be important for Pkd1l1-Pkd2 binding.

What are the technical considerations for using PKD1L1 antibodies in hepatobiliary research?

When investigating PKD1L1 in hepatobiliary development:

• Use antibodies compatible with 2F11 co-staining for biliary epithelial cell identification

• Consider fixation protocols carefully - the search results indicate successful staining after 4% paraformaldehyde fixation followed by methanol post-fixation

• For zebrafish studies, collagenase treatment (1 mg/ml) for 15 minutes helps with antibody penetration

The biliary tree architecture can be assessed by combining PKD1L1 antibody staining with measurements of intrahepatic duct length, liver area, and biliary epithelial cell counts using imaging software like ImageJ . In zebrafish models, PKD1L1 loss leads to developmental biliary anomalies that can be quantified using these techniques.

How should researchers interpret contradictory PKD1L1 antibody staining patterns across different studies?

Discrepancies in PKD1L1 staining patterns may arise from:

• Use of antibodies targeting different protein domains (N-terminal vs. C-terminal)

• Detection of different protein isoforms or proteolytically processed fragments

• Developmental stage-specific expression patterns

• Species-specific differences in protein localization

When encountering contradictory results, consider:

• Validating with multiple antibodies targeting different PKD1L1 epitopes

• Confirming antibody specificity with genetic approaches (siRNA knockdown, CRISPR knockout)

• Verifying developmental timing, as PKD1L1 function changes during embryogenesis

• Consulting mRNA expression data to correlate with protein detection

How can PKD1L1 antibodies contribute to understanding laterality defects?

PKD1L1 plays a crucial role in left-right patterning, with mutations causing laterality defects . When using antibodies to study these defects:

• Examine PKD1L1 localization in the embryonic node/Kupffer's vesicle

• Assess co-localization with Nodal pathway components in left lateral plate mesoderm

• Compare wild-type expression patterns with those in laterality mutants

In zebrafish models, only 18% of pkd1l1 hsc117 mutants showed normal left-sided expression of lefty1/2, with 22% showing right-sided expression and 30% showing bilateral expression . Similarly, heart looping was abnormal in 90% of these mutants. Antibody studies can help determine whether protein mislocalization contributes to these phenotypes.

What approaches can researchers use to investigate PKD1L1 mutations with antibodies?

For studying PKD1L1 mutations:

• Use domain-specific antibodies to determine if mutations affect protein stability or localization

• Compare wild-type and mutant protein expression patterns in affected tissues

• Assess the impact of mutations on PKD1L1-PKD2 interaction using co-immunoprecipitation

The Pkd1l1 rks mutation (D411G) affects a highly conserved region within the second PKD domain, potentially destabilizing the protein structure . Researchers should select antibodies that can detect both wild-type and mutant proteins to determine whether mutations affect expression, localization, or stability.

What are the optimal fixation and immunostaining protocols for PKD1L1 antibodies in different tissues?

Based on successful approaches in the literature:

• For zebrafish larvae: Fix with 4% paraformaldehyde overnight, followed by 100% ice-cold methanol for storage; rehydrate through decreasing methanol concentrations in PBS with 0.2% Triton X-100; perform collagenase treatment (1 mg/ml) for 15 minutes

• For mouse embryonic tissues: Standard paraformaldehyde fixation followed by cryosectioning works well for node tissue

• For cultured cells: 4% paraformaldehyde for 15-20 minutes at room temperature, followed by 0.1-0.2% Triton X-100 permeabilization

Blocking with 10% bovine serum albumin with 0.2% Triton X-100 for 1 hour at room temperature is recommended before antibody incubation overnight at 4°C .

How can researchers quantitatively analyze PKD1L1 expression in developmental studies?

For quantitative analysis of PKD1L1 expression:

• Confocal microscopy with z-stack imaging enables 3D visualization of protein localization

• Process and analyze confocal images using software like Imaris for 3D rendering

• Measure parameters such as:

  • Intrahepatic duct length

  • Liver area

  • Number of biliary epithelial cells

  • Staining intensity normalized to tissue area

When comparing wild-type and mutant samples, ensure consistent imaging parameters and analyze at least 10-15 embryos per genotype for statistical significance.

How can PKD1L1 antibodies be applied in studying biliary atresia and related disorders?

PKD1L1 has emerged as a candidate gene for biliary atresia splenic malformation (BASM) . Researchers can:

• Compare PKD1L1 expression patterns between normal and BASM patient liver samples

• Investigate co-localization with other biliary markers in developmental studies

• Track PKD1L1 expression during biliary tree formation in animal models

Previous research has shown that PKD1L1 expression is weak or absent in liver tissue from BASM patients compared to strong expression in unaffected infant liver tissue . This suggests antibody-based detection could potentially serve as a diagnostic marker for biliary disorders with a genetic basis in PKD1L1 dysfunction.

What are the considerations for using PKD1L1 antibodies in multi-color immunofluorescence studies?

When designing multi-color staining experiments:

• Ensure compatibility of fixation protocols for all target proteins

• Select primary antibodies from different host species to avoid cross-reactivity

• Consider the following validated combinations:

  • PKD1L1 with 2F11 (biliary epithelial marker)

  • PKD1L1 with PKD2 (interaction partner)

  • PKD1L1 with ciliary markers (localization studies)

For hepatobiliary research, combining PKD1L1 staining with 2F11 antibody enables visualization of biliary architecture while assessing PKD1L1 expression in the same structures .