KIF7 Monoclonal Antibody

What is KIF7 and why is it important in cellular biology?

KIF7 is a kinesin motor protein that functions as a critical regulator of the Hedgehog (Hh) signaling pathway. It plays dual roles as both a negative and positive regulator of sonic hedgehog (Shh) and Indian hedgehog (Ihh) pathways, acting downstream of the Smoothened (SMO) receptor through both Suppressor of Fused (SUFU)-dependent and -independent mechanisms . KIF7 is essential for:

• Proper organization of the ciliary tip

• Control of ciliary localization of SUFU-GLI2 complexes

• Localization of GLI3 to cilia in response to Shh

• Regulation of microtubular dynamics

• Prevention of inappropriate activation of transcriptional activator GLI2 in the absence of ligand

• Prevention of the processing of GLI3 into its repressor form

The importance of KIF7 is underscored by the serious developmental consequences associated with its mutation, including Acrocallosal Syndrome, Joubert Syndrome, and intellectual disability with corpus callosum dysgenesis .

What applications are KIF7 monoclonal antibodies suitable for?

KIF7 monoclonal antibodies have been validated for multiple research applications, providing reliable detection of endogenous KIF7 proteins. Based on available information, these antibodies are suitable for:

Each application requires optimization of antibody concentration, incubation times, and detection methods for specific experimental conditions .

How does KIF7 function in Hedgehog signaling pathway regulation?

KIF7 serves as a bifunctional regulator of the Hedgehog signaling pathway, with distinct actions depending on pathway activation status:

In the absence of Hedgehog signaling:

• KIF7 and suppressor of fused (SUFU) restrain Gli transcription factors in the cytosol

• KIF7 promotes Gli3 processing into its transcriptional repressor form

• KIF7 prevents inappropriate activation of the transcriptional activator GLI2

In the presence of Hedgehog signaling:

• KIF7, SUFU, and Gli transcription factors are enriched at the tip of cilium

• KIF7 is required for localization of GLI3 to cilia in response to Shh

• KIF7 prevents the processing of the transcription factor GLI3 into its repressor form

• In keratinocytes, KIF7 promotes the dissociation of SUFU-GLI2 complexes, GLI2 nuclear translocation and Shh signaling activation

This dual regulatory function makes KIF7 a critical control point in Hedgehog signaling, with significant implications for development and disease processes.

What are the recommended storage conditions for KIF7 monoclonal antibodies?

To maintain optimal antibody performance, researchers should follow these storage guidelines for KIF7 monoclonal antibodies:

• Long-term storage: Store at -20°C for up to one year

• Short-term storage: For frequent use, store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles as they can degrade antibody quality and performance

• Most commercial KIF7 antibodies are supplied in a stabilized liquid form containing preservatives such as 50% glycerol, 0.5% BSA, and 0.02% sodium azide

Proper aliquoting upon first thaw is recommended to minimize freeze-thaw cycles and maintain antibody integrity throughout the research project.

How does KIF7's microtubule binding affect ciliary localization and what are the implications for experimental design?

Research has revealed complex relationships between KIF7's microtubule binding properties and its ciliary localization that should be considered when designing experiments:

Key findings on microtubule binding and ciliary localization:

• KIF7's microtubule binding is dispensable for its Hedgehog-induced increase in ciliary localization

• The KIF7 noMT variant (deficient in microtubule binding) can still localize to the cilium tip in response to Hedgehog stimulation (51.4% of SAG-treated cells vs. 14.1% of unstimulated cells)

• KIF7 rigor variant (enhanced microtubule binding) shows increased ciliary tip localization even without Hedgehog stimulation (68.3% of unstimulated cells)

• Motile KIF7 variants (KIF7 motile_21A and KIF7 motile_5C) demonstrated strong localization to the primary cilium tip without Hedgehog stimulation

Experimental design implications:

• When studying Hedgehog pathway activation, researchers should consider that KIF7 localization may be regulated by mechanisms beyond simple microtubule binding

• IFT (intraflagellar transport) appears to be the primary mechanism for Hedgehog-induced localization of KIF7 to the cilium tip

• When using KIF7 mutants, researchers should carefully characterize their microtubule binding properties and ciliary localization patterns

• Experiments investigating KIF7 function should include appropriate controls for both microtubule binding and Hedgehog pathway activation status

This understanding is critical for interpreting results in studies examining ciliary trafficking, Hedgehog signaling, and KIF7 function.

What are the key considerations when using KIF7 monoclonal antibodies in complex tissue samples?

When working with complex tissue samples, researchers should consider several factors to ensure reliable and specific detection of KIF7:

Tissue-specific expression patterns:

• KIF7 expression varies across tissues, with notable presence in developing neural tissues, lung tissues, and T-cell populations

• For developmental studies, temporal expression patterns must be considered, as KIF7 plays crucial roles in tissue patterning and proliferation control

Potential cross-reactivity considerations:

• KIF7 belongs to the kinesin family, which includes structurally similar motor proteins

• Validate antibody specificity using appropriate controls:

  • KIF7 knockout/knockdown tissues

  • Competitive blocking with immunizing peptide

  • Comparison with alternative KIF7 antibody clones

Optimized tissue preparation for KIF7 detection:

• Fixation methods significantly impact epitope accessibility

• Antigen retrieval techniques may be necessary (particularly for formalin-fixed tissues)

• Permeabilization conditions should be optimized for nuclear vs. cytoplasmic detection

For lung tissue specifically, researchers have observed that KIF7 depletion leads to increased cellular density and altered differentiation of respiratory epithelium , suggesting careful consideration of developmental stage when studying KIF7 in this context.

How can researchers effectively study KIF7 mutations and their impact on Hedgehog signaling?

Several methodological approaches can be employed to study KIF7 mutations and their effects on Hedgehog signaling:

Cellular models for studying KIF7 mutations:

• MEF systems: Mouse embryonic fibroblasts from KIF7 knockout mice provide a valuable system for structure-function studies

  • KIF7−/− MEFs show increased growth rates and higher saturation density

  • These cells fail to accumulate Gli2 or Gli3 at the tip of the cilium in response to Hedgehog stimulation

• Patient-derived cells: For clinical mutations, cells derived from patients with KIF7 mutations offer insights into pathophysiology

  • Clinically relevant mutations span a spectrum from severe Acrocallosal Syndrome to milder intellectual disability

Molecular approaches for analyzing signaling alterations:

• Quantitative RT-PCR: Measure expression of Hedgehog target genes such as Ptch1

  • KIF7−/− cells show higher baseline Ptch1 expression but may be refractory to Hedgehog stimulation

• Immunofluorescence analysis: Assess ciliary localization of Gli proteins

  • Expression of KIF7 rigor or motile variants results in Gli2/Gli3 localization to the cilium tip even without Hedgehog stimulation

• Nuclear fractionation and Western blot: Evaluate nuclear translocation of Gli transcription factors

  • KIF7-depleted cells show elevated GLI1 expression in nuclear fractions

Functional readouts:

• Cell proliferation assays (KIF7 mutants show altered growth properties)

• Cyclin D1 expression analysis (increased in KIF7-depleted cells)

• Assessment of microtubule stability markers (acetylated alpha tubulin and detyrosinated alpha tubulin)

These approaches enable comprehensive analysis of how specific KIF7 mutations impact Hedgehog pathway regulation and downstream cellular processes.

What are the technical challenges in detecting KIF7 by Western blot and how can they be addressed?

Detecting KIF7 by Western blot presents several technical challenges due to its size, expression level, and biochemical properties:

Common challenges:

• High molecular weight: KIF7 has a calculated molecular weight of approximately 150.6 kDa, which can make transfer efficiency problematic

• Variable expression levels: Endogenous KIF7 may be expressed at low levels in some cell types

• Protein degradation: As a large protein, KIF7 may be susceptible to proteolytic degradation during sample preparation

• Post-translational modifications: Phosphorylation or other modifications may affect antibody recognition

Optimized Western blot protocol for KIF7 detection:

Validation approaches:

• Include KIF7-depleted or knockout samples as negative controls

• Consider using recombinant KIF7 protein as a positive control

• Verify results with multiple antibody clones when possible

This optimized protocol addresses the technical challenges associated with KIF7 detection by Western blot, enhancing reliability and reproducibility of results.

How do KIF7 functions differ between ciliated and non-ciliated cells, and what implications does this have for experimental design?

KIF7 has distinct functions in ciliated versus non-ciliated cells, which has important implications for experimental design:

Functions in ciliated cells:

• Regulates Hedgehog signaling through control of ciliary trafficking

• Organizes the cilium tip and influences cilium length

• Controls ciliary localization of SUFU-GLI complexes

• Affects IFT-dependent transport within cilia

Functions in non-ciliated cells:

• May regulate microtubule dynamics in the cytoplasm

• Influences cell cycle progression and proliferation

• Affects T-cell development independently of canonical ciliary functions

• May have roles in cytoskeletal organization

Experimental design considerations:

Critical experimental controls:

• Cell type-appropriate positive controls for KIF7 function

• Confirmation of ciliary status in experimental cells

• Assessment of Hedgehog pathway responsiveness in the model system

The distinct functions of KIF7 in different cellular contexts necessitate careful experimental design and interpretation, especially when comparing results across cell types or when studying novel KIF7 functions.