NCKAP1L Antibody

What is NCKAP1L and why is it an important research target?

NCKAP1L (NCK-associated protein 1-like), also known as HEM1, is a membrane-associated protein belonging to the HEM family of tissue-specific transmembrane proteins that are highly conserved from invertebrates through mammals. It functions primarily as a hematopoietic cell-specific actin regulatory protein and is a critical component of the WAVE regulatory complex (WRC), which signals downstream of activated Rac to stimulate F-actin polymerization in response to immunoreceptor signaling . The importance of NCKAP1L lies in its essential role in reorganization of the actin cytoskeleton in immune cells, making it a valuable target for immunological research . Recent studies have also revealed unexpected roles in biliary epithelial cells through a previously uncharacterized minor splice isoform , suggesting broader physiological importance than previously recognized.

What applications are NCKAP1L antibodies suitable for?

NCKAP1L antibodies have been validated for multiple experimental applications. Based on the available data, these antibodies can be effectively used in:

When selecting an NCKAP1L antibody for a specific application, researchers should verify the validation status for their particular experimental system and consider the recommended working dilutions provided by manufacturers .

What is the species reactivity of commonly available NCKAP1L antibodies?

Different NCKAP1L antibodies show varied species reactivity profiles. Based on available data:

What are the optimal protocols for using NCKAP1L antibodies in immunohistochemistry?

For successful immunohistochemistry using NCKAP1L antibodies, the following protocol has shown reliable results:

• Tissue preparation: Fix tissues in 2% formaldehyde in PEM (0.1 M Pipes, 2 mM EGTA, and 1.0 mM MgSO4). For human samples, formalin-fixed paraffin-embedded (FFPE) tissue sections are commonly used .

• Antigen retrieval: This step is critical for FFPE tissues to expose epitopes masked during fixation. Heat-induced epitope retrieval in citrate buffer (pH 6.0) is generally effective.

• Blocking: Use 10% Triton X-100 and 20% fetal bovine serum (FBS) in phosphate-buffered saline (PBS) for blocking non-specific binding sites .

• Primary antibody incubation: For antibody ab272631, a dilution of 1:500 has been successfully used. For HPA039490, dilutions between 1:200-1:500 are recommended .

• Detection system: Use appropriate secondary antibodies such as goat anti-rabbit IgG conjugated with fluorophores like ALEXA FLUOR 568 at a 1:200 dilution .

• Controls: Always include positive controls (tissues known to express NCKAP1L, such as lymph tissue, tonsil, or liver) and negative controls (primary antibody omitted) to validate staining specificity .

This protocol has successfully demonstrated NCKAP1L expression in various tissues, including biliary epithelial cells and vascular endothelial cells, revealing previously unknown expression patterns .

How should Western blotting protocols be optimized for NCKAP1L detection?

NCKAP1L has a calculated molecular weight of 128 kDa, which requires specific considerations for optimal detection by Western blotting:

• Sample preparation: Use NETN lysis buffer for effective protein extraction from tissues and cell lines. For tissues with high protease activity, adding protease inhibitors is crucial .

• Gel electrophoresis: Use lower percentage (6-8%) SDS-PAGE gels for better resolution of high molecular weight proteins like NCKAP1L.

• Transfer conditions: Perform wet transfer at lower amperage (250-300 mA) for longer duration (90-120 minutes) to ensure complete transfer of large proteins.

• Blocking: 5% non-fat dry milk or BSA in TBST has been effective.

• Antibody incubation: For optimal results:

  • Ab226302: Use at 0.1 μg/mL

  • 10454-1-AP: Follow manufacturer's recommended dilution

  • HPA039490: Use at 0.04-0.4 μg/mL

• Detection: Enhanced chemiluminescence (ECL) with appropriate exposure times (10 seconds has been reported as effective for some antibodies) .

• Expected results: Verify bands at the predicted molecular weight of 128 kDa. NCKAP1L has been successfully detected in human cell lines (HeLa, HEK-293T, Jurkat) and mouse cell lines (TCMK-1, NIH/3T3) .

For difficult samples, consider using immunoprecipitation to enrich for NCKAP1L protein before Western blotting to enhance detection sensitivity .

How can NCKAP1L antibodies be used to distinguish between different splice variants?

Recent research has identified a previously unannotated minor splice isoform of NCKAP1L that plays a crucial role in biliary epithelial cells . To distinguish between splice variants:

• Epitope mapping: Determine whether your antibody's epitope is present in all splice variants. The minor splice isoform identified in zebrafish has a different C-terminus from the primary isoform. Antibodies targeting the N-terminal region (like ab226302) may detect both isoforms, while C-terminal antibodies might be isoform-specific .

• Multiple antibody approach: Use antibodies targeting different epitopes in parallel experiments to identify potential isoform-specific patterns.

• Molecular weight analysis: Carefully analyze Western blot results for bands of different molecular weights that may correspond to different isoforms. The minor splice isoform may appear as a lower molecular weight band.

• Controls: Use tissues known to express specific isoforms as positive controls. Based on current research, hematopoietic tissues predominantly express the primary isoform, while liver tissue may express both the primary and minor isoforms .

• RT-PCR validation: Complement antibody-based detection with RT-PCR using isoform-specific primers to confirm the presence of specific splice variants at the mRNA level.

The zebrafish research demonstrating that the minor splice isoform of NCKAP1L plays a role in biliary epithelial cells suggests that similar minor isoforms might exist in humans. When studying NCKAP1L in non-hematopoietic tissues, particularly in the liver, researchers should be aware of potential splice variants and design experiments accordingly .

What are the known challenges in detecting NCKAP1L in tissue samples?

Detecting NCKAP1L in tissue samples presents several challenges that researchers should anticipate:

• Tissue-specific expression levels: NCKAP1L is predominantly expressed in hematopoietic cells, making detection in other tissues challenging due to lower expression levels. In biliary epithelial cells, NCKAP1L was observed as small puncta rather than strong diffuse staining, requiring high-sensitivity detection methods .

• Protein degradation: As a component of protein complexes, NCKAP1L stability may depend on interaction partners. In zebrafish nckap1l mutants, the protein was degraded in biliary epithelial cells, suggesting that disruption of the WAVE complex can lead to protein degradation .

• Distinguishing from NCKAP1: NCKAP1L shares significant homology with NCKAP1 (also known as NAP1). Ensure your antibody specifically recognizes NCKAP1L without cross-reactivity to NCKAP1 .

• Isoform-specific detection: As discussed, different splice variants may be expressed in different tissues, requiring careful antibody selection.

• Fixation sensitivity: The epitope recognized by some NCKAP1L antibodies may be sensitive to fixation conditions. In particular, researchers studying zebrafish found that careful fixation in 2% formaldehyde in PEM buffer was effective for immunohistochemistry .

To overcome these challenges, researchers should:

• Use positive control tissues known to express NCKAP1L (lymph nodes, tonsil)

• Optimize fixation and antigen retrieval conditions

• Consider signal amplification methods for tissues with low expression

• Validate findings with complementary approaches (RT-PCR, RNA-seq)

How do NCKAP1L antibodies perform in studying WAVE complex dynamics?

NCKAP1L is a critical component of the WAVE regulatory complex (WRC), making NCKAP1L antibodies valuable tools for studying this complex's dynamics:

• Co-immunoprecipitation studies: NCKAP1L antibodies can be used to pull down the entire WAVE complex. In zebrafish studies, immunostaining for other WAVE complex components (WAVE1, Abi1, HSPC300) showed that these proteins are degraded in nckap1l mutants, indicating that NCKAP1L is essential for complex stability in hematopoietic and biliary epithelial cells .

• Cellular localization: Immunofluorescence with NCKAP1L antibodies can reveal the subcellular localization of the WAVE complex during cellular processes like migration, phagocytosis, and immune synapse formation.

• Activation dynamics: By combining NCKAP1L antibodies with phospho-specific antibodies against other complex components, researchers can study the activation state of the complex.

• Interaction with regulatory pathways: NCKAP1L antibodies have been used to demonstrate that NCKAP1L genetically interacts with the Cdk5 and Rac1 pathways in biliary epithelial cells, suggesting that NCKAP1L might be a signaling hub integrating multiple pathways .

• Tissue-specific complex composition: Different tissues may have different WAVE complex compositions. NCKAP1L antibodies can help determine whether NCKAP1L-containing complexes are functionally distinct from NCKAP1-containing complexes.

When studying WAVE complex dynamics, consider using dual immunostaining for NCKAP1L and other complex components (WAVE1, WAVE2, Abi1, HSPC300) to assess complex integrity and localization in different experimental conditions .

How are NCKAP1L antibodies used in studying immune system disorders?

NCKAP1L plays crucial roles in immune cell function, making its antibodies valuable tools for studying immune disorders:

• Wiskott-Aldrich syndrome research: NCKAP1L has been connected to Wiskott-Aldrich syndrome, where dysfunction is often tied to aberrant actin dynamics involving the WAS protein. NCKAP1L antibodies can help elucidate the relationship between NCKAP1L and WAS protein in patient samples .

• T-cell function studies: NCKAP1L is required for proper mechanistic target of rapamycin complex 2 (mTORC2)-dependent AKT phosphorylation, cell proliferation, and cytokine secretion in T-cells. Antibodies can track NCKAP1L localization during T-cell activation and at the immunological synapse .

• Neutrophil migration analysis: NCKAP1L is essential for efficient neutrophil migration. Antibodies can be used to study NCKAP1L's role in neutrophil chemotaxis and actin dynamics during migration .

• Immune cell development tracking: NCKAP1L controls lymphocyte development, activation, proliferation, and homeostasis. Antibodies can be used to track NCKAP1L expression during different stages of immune cell development and maturation.

When studying immune disorders, it's important to use appropriate experimental controls and to consider that alterations in NCKAP1L may affect multiple aspects of immune cell function due to its role in actin cytoskeleton regulation .

What evidence exists for NCKAP1L's role in cancer, and how can antibodies help investigate this?

Emerging evidence suggests potential roles for NCKAP1L in cancer:

• Osteosarcoma metastasis: NCKAP1L has been identified as a potential predictive marker for osteosarcoma metastasis, suggesting it may play a role in cancer cell migration or invasion .

• Tumor microenvironment studies: Given NCKAP1L's role in immune cell function, antibodies can be used to study how immune cells in the tumor microenvironment might be affected by alterations in NCKAP1L expression.

• Cancer cell migration analysis: NCKAP1L antibodies can track the protein's involvement in cancer cell migration through its effects on the actin cytoskeleton.

• Immune surveillance: NCKAP1L's role in immune cell function suggests it might be important for immune surveillance against cancer. Antibodies can help study this aspect in tumor models.

When investigating NCKAP1L in cancer, researchers should:

• Compare expression levels between normal and malignant tissues

• Correlate expression with clinical outcomes

• Investigate potential associations with metastatic potential

• Consider both tumor cell-intrinsic effects and effects on tumor-infiltrating immune cells

How are NCKAP1L antibodies contributing to vascular disease research?

Recent research has implicated NCKAP1L in vascular diseases, particularly abdominal aortic aneurysms (AAA):

• Expression in AAA tissues: NCKAP1L was found to be robustly expressed in AAA tissues, and in the H2O2-induced vascular smooth muscle cell (VSMC) model, increased expression of NCKAP1L was also observed .

• Regulatory network analysis: NCKAP1L has been identified as part of a regulatory network involving the long non-coding RNA PVT1 and microRNA miR-3127-5p. This network appears to modulate VSMC proliferation, apoptosis, and inflammation in AAA pathogenesis .

• Mechanistic studies: NCKAP1L antibodies can be used to track protein expression changes in response to treatments targeting the PVT1/miR-3127-5p/NCKAP1L axis, helping to elucidate the underlying mechanisms of AAA.

• Vascular cell phenotyping: Given NCKAP1L's role in actin dynamics, antibodies can help characterize how changes in NCKAP1L expression affect VSMC phenotype, migration, and response to inflammatory stimuli.

For researchers studying NCKAP1L in vascular diseases, it may be beneficial to:

• Compare expression patterns in different vascular beds

• Investigate potential splice variant-specific effects

• Study the interaction between NCKAP1L-expressing vascular cells and immune cells

• Develop experimental models where NCKAP1L expression can be manipulated specifically in vascular cells

What are the most effective methods for validating NCKAP1L antibody specificity?

Validating antibody specificity is crucial for reliable experimental results. For NCKAP1L antibodies, consider these approaches:

• Genetic knockout controls: Use CRISPR/Cas9-edited cell lines lacking NCKAP1L expression as negative controls. In zebrafish research, CRISPR/Cas9-mediated NCKAP1L deletion confirmed the specificity of antibody staining patterns .

• Knockdown experiments: siRNA or shRNA-mediated knockdown of NCKAP1L should result in reduced antibody signal.

• Peptide competition: Pre-incubation of the antibody with the immunizing peptide should block specific binding and eliminate the signal.

• Multiple antibody comparison: Use antibodies targeting different epitopes of NCKAP1L and compare staining patterns.

• Cross-reactivity testing: Test the antibody against recombinant NCKAP1 (the closest homolog) to ensure it doesn't cross-react.

• Immunoprecipitation followed by mass spectrometry: This approach can confirm that the antibody is pulling down NCKAP1L rather than other proteins.

• Correlation with mRNA expression: Compare protein detection patterns with known mRNA expression patterns from databases or qPCR analysis.

The HPA039490 antibody, for instance, has been tested on protein arrays of 364 human recombinant protein fragments to verify specificity , providing a high level of confidence in its specificity.

What storage and handling conditions are optimal for maintaining NCKAP1L antibody performance?

Proper storage and handling of NCKAP1L antibodies is essential for maintaining their performance over time:

Note that some specialized formulations may have different requirements. For instance, the information for 10454-1-AP indicates that 20μl sizes contain 0.1% BSA , which may affect stability and storage recommendations.

What are the key differences between NCKAP1L and NCKAP1 antibodies that researchers should be aware of?

NCKAP1L and NCKAP1 (also known as NAP1) are homologous proteins that function in different cell types as part of the WAVE complex. Understanding the differences between antibodies against these proteins is important:

• Cellular expression patterns:

  • NCKAP1L is predominantly expressed in hematopoietic cells and, as recently discovered, in biliary epithelial cells .

  • NCKAP1 is more broadly expressed across various tissues and cell types .

• Epitope selection:

  • Anti-NCKAP1 antibodies (like ab226302) often target the N-terminal region of the protein .

  • NCKAP1L antibodies may target various regions; it's important to check the specific epitope.

• Molecular weight:

  • Both proteins have similar calculated molecular weights (approximately 128 kDa) .

  • This similarity makes it crucial to ensure antibody specificity to distinguish between them.

• Cross-reactivity potential:

  • Due to sequence homology, there's potential for cross-reactivity.

  • Validation using specific controls (like knockout cells for each protein) is essential.

• Functional applications:

  • NCKAP1L antibodies are particularly valuable for studying immune cell functions and newly discovered roles in biliary cells .

  • NCKAP1 antibodies are useful for studying broader cellular processes like lamellipodia formation and BDNF-NTRK2 endocytic trafficking .

• Species considerations:

  • Available NCKAP1L antibodies have been validated primarily in human, mouse, and rat samples .

  • NCKAP1 antibodies may have different species reactivity profiles.

When designing experiments involving these related proteins, researchers should carefully select antibodies with validated specificity for their target of interest and consider including controls that can distinguish between NCKAP1L and NCKAP1.

What emerging applications of NCKAP1L antibodies show particular promise?

Several emerging applications of NCKAP1L antibodies show significant promise for advancing our understanding of cellular processes and disease mechanisms:

• Single-cell analysis of immune cell heterogeneity: NCKAP1L antibodies could be used in mass cytometry (CyTOF) or imaging mass cytometry to characterize immune cell subpopulations based on NCKAP1L expression levels and localization patterns.

• Studying non-canonical functions: The discovery of NCKAP1L's role in biliary epithelial cells opens up possibilities for identifying other non-hematopoietic cell types where NCKAP1L may play important roles . Antibodies will be crucial for these investigations.

• Therapeutic target validation: As our understanding of NCKAP1L's role in various diseases grows, antibodies will be essential for validating it as a potential therapeutic target and for monitoring changes in its expression or localization in response to experimental therapeutics.

• Biomarker development: The potential link between NCKAP1L and diseases like osteosarcoma metastasis and abdominal aortic aneurysms suggests that NCKAP1L antibodies might be valuable for developing diagnostic or prognostic biomarkers.

• Structural studies: Using antibodies that recognize specific conformational states of NCKAP1L could help elucidate how its structure changes during WAVE complex activation.

• Developmental biology: Given the role of actin dynamics in development, NCKAP1L antibodies could help track the protein's expression and function during embryonic and postnatal development of hematopoietic and biliary systems.

These emerging applications highlight the importance of continuing to develop and characterize highly specific NCKAP1L antibodies for diverse experimental techniques.

How might NCKAP1L antibodies contribute to understanding the role of the WAVE complex in non-immune cells?

The recent discovery of NCKAP1L's role in biliary epithelial cells challenges the previous understanding that NCKAP1L is exclusively a hematopoietic cell-specific protein . This finding opens up new avenues for research where NCKAP1L antibodies can contribute to understanding the WAVE complex in non-immune cells:

• Tissue-specific WAVE complex composition: NCKAP1L antibodies can help determine whether the composition of the WAVE complex differs between immune and non-immune cells, potentially explaining functional differences.

• Developmental processes: In zebrafish, NCKAP1L was found to be essential for branching morphogenesis of the intrahepatic biliary network . Antibodies can help track NCKAP1L expression during organ development across different species.

• Splice variant function: The minor splice isoform of NCKAP1L discovered in biliary epithelial cells suggests that similar isoforms might exist in other non-immune cells. Isoform-specific antibodies could help identify these variants and their functions.

• Pathological conditions: NCKAP1L's role in biliary cells suggests potential involvement in cholangiopathies. The zebrafish research noted that "sequence data derived from patients with biliary atresia and other cholangiopathies should be reexamined focusing on mutations in the last exon of human NCKAP1L" .

• Genetic interactions: NCKAP1L was found to genetically interact with the Cdk5 and Rac1 pathways in biliary epithelial cells . Antibodies can help investigate whether similar interactions exist in other cell types.

• Comparative studies: Using antibodies to compare NCKAP1L and NCKAP1 expression in various tissues could reveal patterns that help explain why some cells utilize NCKAP1L while others use NCKAP1.

These research directions highlight how NCKAP1L antibodies are essential tools for expanding our understanding of actin cytoskeleton regulation beyond the immune system.

What are the recommended positive and negative controls for NCKAP1L antibody experiments?

Based on the available research, the following controls are recommended for NCKAP1L antibody experiments:

Positive Controls (Tissues/Cells with known NCKAP1L expression):

• Human lymph tissue

• Human tonsil tissue

• Human liver tissue (particularly for examining biliary epithelial cells)

• Jurkat cells (human T cell leukemia cell line)

• Primary immune cells (macrophages, neutrophils, T cells)

• Vascular endothelial cells

Negative Controls:

• Primary antibody omission control

• NCKAP1L knockout or knockdown cells/tissues

• Non-immune tissues with minimal hematopoietic cell infiltration (validated by absence of immune cell markers)

• Peptide competition control (pre-incubation of antibody with immunizing peptide)

• Isotype control antibody

Additional Control Considerations:

• For splice variant studies, controls should include tissues known to express specific variants

• When studying disease states, matched normal tissue should be included

• For cell type-specific expression, co-staining with cell type markers is recommended

• When comparing NCKAP1L with NCKAP1, include tissues known to express only one of these proteins

Proper controls are essential for interpreting NCKAP1L antibody results, especially given its expression in specific cell types and potential splice variants .

What information should researchers include in publications when reporting NCKAP1L antibody use?

To ensure reproducibility and transparency in research using NCKAP1L antibodies, publications should include:

• Antibody identification:

  • Complete catalog number (e.g., 10454-1-AP, HPA039490, ab272631)

  • Manufacturer/source

  • Clone number for monoclonal antibodies

  • Host species and antibody type (polyclonal/monoclonal)

  • Lot number (especially important for polyclonal antibodies)

• Epitope information:

  • Target region of NCKAP1L (N-terminal, C-terminal, specific amino acid range)

  • Immunogen sequence if available (e.g., "VLIRMYNIKKTCSDPKSKPPFLLEKSMEPSLKYINKKFPNIDVRNSTQHLGPVHREKAEIIRFLTNYYQSFVD" for HPA039490)

  • Information on which splice variants the antibody should recognize

• Validation methods:

  • Description of how specificity was confirmed

  • References to previous validations

  • Results of control experiments

• Experimental details:

  • Complete protocol including buffers, incubation times, temperatures

  • Dilution or concentration used

  • Antigen retrieval method for IHC/IF

  • Detection system (secondary antibody details, visualization method)

  • Sample preparation details

• Imaging/detection parameters:

  • For microscopy: exposure times, microscope settings

  • For Western blotting: exposure time, molecular weight markers, loading controls

• Controls used:

  • Positive and negative controls

  • Validation in knockout/knockdown samples if available