KANK1 Antibody

What applications are KANK1 antibodies validated for?

KANK1 antibodies are validated for multiple applications including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF/IF-P), Immunoprecipitation (IP), and ELISA. According to validation data, specific antibodies such as 29782-1-AP have been confirmed effective in these applications with standardized protocols . Flow cytometry (FC) has also been validated for certain KANK1 antibody products, particularly when analyzing intracellular expression .

What is the expected molecular weight of KANK1 in Western blot analysis?

KANK1 typically appears between 130-200 kDa in Western blot analysis . The calculated molecular weight is 147 kDa, but post-translational modifications can cause variation in observed molecular weight . Two alternatively spliced isoforms (KANK1-L at ~175 kDa and KANK1-S at ~160 kDa) are present in many human tissues, which may account for multiple bands in Western blot analysis .

What species reactivity has been confirmed for KANK1 antibodies?

Most commercially available KANK1 antibodies demonstrate reactivity with human and mouse samples . Some antibodies also show reactivity with rat samples . When selecting an antibody for cross-species studies, it's important to verify the tested reactivity information provided by manufacturers. The homology between species should be considered when interpreting results from non-validated species.

How should I design experiments to study KANK1 interactions with focal adhesion components?

When studying KANK1 interactions with focal adhesion components, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): Use antibodies against KANK1 to pull down protein complexes, then probe for talin, liprin-β1, or other suspected binding partners. Research has confirmed that endogenous KANK1 specifically co-precipitates with BIG1 antibodies but not BIG2 .

• Deletion mapping: Create truncated KANK1 constructs to identify specific domains required for interactions. The KN domain (residues 30-68) has been shown to strongly and specifically accumulate within focal adhesions .

• Proximity assays: Consider using techniques like FRET, BiFC, or proximity ligation assays to detect in situ interactions.

• Structural analysis: When studying direct interactions, consider using structural methods like the HADDOCK docking program that was used to model KANK1/R7 complex interactions .

• Mutational analysis: Point mutations in binding domains can validate key residues for protein-protein interactions. For example, the KANK1-4A mutant disrupts binding to talin while maintaining liprin-β1 interaction .

What controls should be included when validating KANK1 antibodies in knockout/knockdown studies?

Properly controlled KANK1 knockdown/knockout studies should include:

• Positive controls: Use cell lines with confirmed KANK1 expression (e.g., HEK-293T cells) to validate antibody specificity .

• Negative controls:

  • Non-targeting siRNA controls to account for transfection effects

  • Secondary antibody-only controls to identify non-specific binding

  • KANK1 knockout/knockdown samples as true negative controls

• Rescue experiments: Re-expression of wild-type KANK1 in knockdown cells should restore normal phenotype and antibody detection .

• Verification methods: Use multiple detection methods (e.g., WB and IF) to confirm knockdown efficiency. Endogenous BIG1, BIG2, KANK1, and KIF21A levels were reported at 14 ± 6%, 9 ± 3%, 14 ± 5%, and 11 ± 2%, respectively, of control cells 48 hours after adding specific siRNAs .

• Quantification: Include densitometric analysis of Western blots with appropriate loading controls.

How can I optimize KANK1 antibody dilutions for different applications?

Optimal dilution varies by application and specific antibody. Follow these recommendations:

For antigen retrieval in IHC:

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

"It is recommended that this reagent should be titrated in each testing system to obtain optimal results" . Sample-dependent variation may require additional optimization.

What are the recommended storage conditions for KANK1 antibodies?

Most KANK1 antibodies should be stored at -20°C for long-term stability. Specific storage recommendations include:

• Store at -20°C in aliquots to minimize freeze-thaw cycles

• Stable for one year after shipment when properly stored

• Some antibodies (e.g., 68613-1-PBS) require storage at -80°C

• Storage buffers typically include PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Small size aliquots (20μl) may contain 0.1% BSA as a stabilizer

Aliquoting is generally recommended for antibodies stored at -20°C, though some manufacturers note it is unnecessary for specific formulations .

How can I use KANK1 antibodies to study its role in cancer development?

KANK1 has been implicated in several cancer types, including breast cancer and oral squamous cell carcinoma (OSCC). When investigating KANK1 in cancer:

• Expression analysis: Compare KANK1 expression between normal and tumor tissues. Studies have shown KANK1 promotes proliferation and survival of PyMT-transformed mammary tumor cells in vivo , while it appears to function as a tumor suppressor in OSCC .

• Mechanistic studies: Investigate KANK1's interaction with key signaling pathways:

  • In breast cancer: KANK1 competes with the tumor suppressor Scribble for NOS1AP binding, reducing Hippo pathway activity, leading to TAZ stabilization and nuclear accumulation .

  • In OSCC: Overexpression of Kank1 decreased proliferation of OSCC cells both in vitro and in vivo by regulating Yap to inhibit proliferation and promote apoptosis .

• Subcellular localization: In transformed mammary epithelial cells, KANK1 localizes to the basal side of basement membrane-attached cells but relocates to cell-cell junctions when cells lose basement membrane contact .

• Clinical correlation: Assess the relationship between KANK1 expression and clinical parameters. In OSCC, poor Kank1 expression correlates with advanced stage and poor prognosis .

• Functional validation: Use KANK1 overexpression or knockdown studies to confirm its role. For example, researchers found that "by overexpression of Kank1, the proliferation ability of the OSCC cells decreased both in vitro and in vivo, the proportion of apoptotic cells increased, and the mitochondrial transmembrane potential decreased" .

What considerations are important when studying KANK1 interactions with the cytoskeleton?

KANK1 plays crucial roles in cytoskeletal regulation, particularly at the interface between focal adhesions and microtubules:

• Dual labeling: Use antibodies against KANK1 alongside cytoskeletal markers (actin, tubulin) and focal adhesion proteins (talin, paxillin) to visualize spatial relationships.

• Live cell imaging: Consider using fluorescently tagged KANK1 constructs to monitor dynamic interactions. Studies using EB3-mRFP in KANK1/2 depleted cells showed that "microtubule plus end growth velocity was almost 2.5 times slower at the cell margin compared to the central part of the cell" .

• Functional domains: Different KANK1 domains mediate specific interactions:

  • The KN domain binds to talin's R7 domain

  • The coiled-coil domain interacts with liprin-β1

  • The ankyrin repeat domain binds to KIF21A

• Subcellular dynamics: KANK1 shows specific localization patterns requiring careful imaging settings:

  • Full-length KANK1 localizes to focal adhesion rims

  • The KN domain alone accumulates within focal adhesions

  • The KANK1-△ANKR mutant is excluded from focal adhesions but accumulates in their vicinity

• Functional outcomes: Measure the effects of KANK1 perturbation on:

  • Microtubule dynamics (growth rate, stability)

  • Cell migration (wound healing assays show BIG1, BIG2, or KANK1 siRNA treatments each delayed wound closure)

  • Focal adhesion turnover

How can I troubleshoot non-specific binding with KANK1 antibodies?

When experiencing non-specific binding with KANK1 antibodies:

• Optimize blocking conditions: Use 5% BSA or 5% non-fat dry milk in TBS-T, with extended blocking times (1-2 hours at room temperature).

• Increase washing steps: Additional and longer washes with TBS-T can reduce background.

• Adjust antibody concentration: Titrate antibody concentration downward if background is high.

• Consider fixation methods: Different fixation protocols may affect epitope accessibility and non-specific binding.

• Validate specificity: Use KANK1 knockdown/knockout samples to confirm band specificity. Some antibodies show multiple bands between 130-200 kDa, and cross-reactivity issues have been reported, as noted: "The lower molecular weight band seen in the immunoblot is thought to be non-specific" .

• Sample preparation: Ensure complete protein denaturation for Western blots and appropriate antigen retrieval for IHC/IF.

What are the limitations of current KANK1 antibodies for distinguishing isoforms?

Current limitations in studying KANK1 isoforms include:

How might KANK1 antibodies be used to explore its role in hematopoietic disorders?

Recent findings suggest potential roles for KANK1 in hematopoietic processes:

• Clinical relevance: A germline loss of heterozygosity mutation encompassing the KANK1 gene was identified in a young patient with myelodysplastic neoplasm (MDS) with no additional disease-related genomic aberrations .

• Animal models: KANK1 knockout mice showed "alteration in the colony forming and proliferative potential of bone marrow (BM) cells and a decrease in hematopoietic stem and progenitor cells (HSPCs) population frequency" , suggesting roles in normal hematopoiesis.

• Marker expression analysis: Comprehensive studies revealed KANK1 involvement in immune cell development regulation .

• Fusion protein significance: A t(5;9) translocation resulting in a PDGFRB-KANK1 fusion protein was detected in a patient with myeloproliferative neoplasm characterized by severe thrombocythemia .

• Experimental approaches: Researchers could use KANK1 antibodies to:

  • Compare expression in normal vs. pathological bone marrow samples

  • Analyze KANK1 expression in sorted hematopoietic cell populations

  • Investigate KANK1 knockout effects on hematopoietic differentiation

  • Study potential KANK1 involvement in cytoskeletal regulation during immune cell development and function

What emerging techniques might enhance the study of KANK1 using antibody-based approaches?

Several cutting-edge approaches could advance KANK1 research:

• Super-resolution microscopy: Techniques like STORM or PALM could better resolve KANK1's distinct localization at focal adhesion rims, which conventional microscopy may not clearly distinguish.

• Proximity proteomics: BioID or APEX2 tagging of KANK1 could identify novel interaction partners beyond the currently known associations with talin, liprin-β1, and KIF21A.

• Single-cell analysis: Combining KANK1 antibodies with single-cell technologies could reveal heterogeneity in expression and function across cell populations.

• CRISPR-based approaches: CRISPR knock-in of tags or fluorescent proteins at the endogenous KANK1 locus could enable studies of native expression levels and dynamics.

• Optogenetics: Light-controlled manipulation of KANK1 localization or interaction could help dissect its dynamic roles in cytoskeletal regulation.

• Intrabodies: Developing intracellular antibodies against KANK1 could enable live-cell tracking and functional perturbation.

• Multiplex imaging: Simultaneous visualization of KANK1 with multiple binding partners could provide contextual information about its function in different subcellular compartments.