KLHL13 Antibody

What is KLHL13 and what are its key structural domains?

KLHL13 (Kelch-like protein 13) belongs to the Kelch-like family of proteins characterized by three distinct domains: a BTB/POZ (bric-a-brac, tramtrack, broad complex/poxvirus and zinc finger) domain, a BACK domain, and a Kelch domain consisting of six Kelch repeats . The BTB domain mediates protein-protein interactions, while the Kelch repeats form a β-propeller structure responsible for substrate recognition and binding . These structural elements enable KLHL13 to function as a substrate-specific adapter protein within larger protein complexes.

What is the primary function of KLHL13 in cellular processes?

KLHL13 functions as a substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex required for mitotic progression and cytokinesis . The BCR(KLHL9-KLHL13) E3 ubiquitin ligase complex specifically mediates the ubiquitination of Aurora B kinase (AURKB) and controls its dynamic behavior on mitotic chromosomes, thereby coordinating faithful mitotic progression and completion of cytokinesis . This ubiquitination is critical for proper chromosome segregation during cell division.

What is the tissue distribution and expression pattern of KLHL13?

KLHL13 is predominantly expressed in brain and testis tissues, with notable expression also detected in the thalamus and whole embryo during development . In zebrafish, KLHL13 is expressed in the olfactory bulb and telencephalon . Understanding this tissue-specific expression pattern is important when designing experiments targeting KLHL13 in different research models.

What criteria should be considered when selecting a KLHL13 antibody for specific applications?

When selecting a KLHL13 antibody, researchers should evaluate:

• Application compatibility: Verify the antibody is validated for your specific application (WB, IHC, IF, FACS, ELISA)

• Host species and isotype: Consider compatibility with your experimental design, especially for multiplexing

• Epitope recognition: N-terminal vs. full-length antibodies may yield different results

• Species reactivity: Confirm the antibody recognizes KLHL13 in your species of interest (human, mouse, rat)

• Validation methods: Prioritize antibodies validated against knockout controls, as recommended by antibody validation protocols

The antibody selection should be guided by the specific requirements of your experiment and the available validation data for each candidate antibody.

How can researchers validate the specificity of KLHL13 antibodies?

The optimal KLHL13 antibody validation methodology involves:

• Using an appropriate wild-type cell and an isogenic CRISPR knockout (KO) version of the same cell as the basis for testing

• Performing western blot analysis to confirm the detection of a band at the predicted molecular weight (74 kDa) or observed molecular weight (68 kDa)

• Confirming the absence of signal in knockout or knockdown cells

• Validating across multiple applications if the antibody will be used in different techniques

• Testing reactivity with recombinant KLHL13 protein as a positive control

This rigorous validation approach ensures antibody specificity and minimizes false positive or negative results in subsequent experiments.

What are the common pitfalls in KLHL13 antibody selection that may affect experimental outcomes?

Several factors can compromise experimental results when working with KLHL13 antibodies:

• Cross-reactivity with other KLHL family members (particularly KLHL9, which shares functional similarities)

• Variation in molecular weight detection (predicted: 74 kDa; observed: 68 kDa)

• Insufficient validation against knockout controls

• Incompatibility between application and antibody optimization

• Buffer composition effects on antibody performance

To mitigate these issues, researchers should perform preliminary validation experiments and include appropriate positive and negative controls in all studies involving KLHL13 antibodies.

What are the optimal conditions for using KLHL13 antibodies in Western blot applications?

For optimal Western blot detection of KLHL13:

• Sample preparation: Use fresh cell lysates from tissues with known KLHL13 expression (brain, testis)

• Protein loading: 20-30 μg of total protein is typically sufficient

• Recommended dilutions: 1:500-1:2000 range, with optimal results at 1:1000

• Expected band size: 68-74 kDa (variations may occur due to post-translational modifications)

• Positive controls: HEK-293T, Jurkat cells, or HeLa cells show detectable levels of endogenous KLHL13

When troubleshooting weak signals, extending primary antibody incubation time (overnight at 4°C) and optimizing blocking reagents may improve detection sensitivity.

How should KLHL13 antibodies be used for immunoprecipitation to study protein-protein interactions?

For effective immunoprecipitation of KLHL13:

• Use antibodies specifically validated for IP applications

• Start with 1-2 mg of total protein lysate

• Pre-clear lysates to reduce non-specific binding

• Consider interaction conditions: KLHL13 interactions with Aurora B and other substrates may be cell cycle-dependent

• Include appropriate controls to verify specificity:

  • IgG control to assess non-specific binding

  • Input samples (5-10%) for comparison

  • If studying KLHL13-CUL3 interactions, consider reciprocal IPs

When analyzing KLHL13 interactions with potential substrates, validation through reverse co-immunoprecipitation approaches is recommended to confirm the specificity of the interaction .

What considerations are important when using KLHL13 antibodies for immunohistochemistry or immunofluorescence?

For successful IHC/IF applications:

• Tissue fixation: Formalin fixation followed by paraffin embedding is compatible with most KLHL13 antibodies

• Antigen retrieval: TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 can be an alternative

• Recommended dilutions: 1:50-1:500 for IHC; 1:200-1:1000 for IF

• Positive control tissues: Brain, skeletal muscle, and lung tissues show detectable KLHL13 expression

• Counterstaining: DAPI for nuclear visualization helps confirm the subcellular localization pattern (KLHL13 shows both cytoplasmic and nuclear distribution)

The subcellular localization of KLHL13 may vary depending on cell cycle stage, with more prominent centromeric/midbody localization during mitosis.

How can KLHL13 antibodies be used to investigate its role in the ubiquitin-proteasome pathway?

To study KLHL13's role in the ubiquitin-proteasome pathway:

• Co-immunoprecipitation studies: Use KLHL13 antibodies to pull down the CUL3-KLHL9-KLHL13 complex and analyze associated proteins

• Ubiquitination assays: Combine KLHL13 immunoprecipitation with ubiquitin antibodies to detect ubiquitinated substrates

• Proteasome inhibition experiments: Compare KLHL13 substrate levels (e.g., Aurora B) with and without proteasome inhibitors (MG132)

• Proximity ligation assays: Visualize KLHL13-substrate interactions in situ

• CRISPR/Cas9 knockout validation: Generate KLHL13-deficient cells to confirm antibody specificity and substrate stabilization

These approaches can reveal novel KLHL13 substrates and regulatory mechanisms in the ubiquitin-proteasome system.

What methods can be used to study KLHL13's interaction with Aurora B in mitotic regulation?

To investigate KLHL13-Aurora B interactions:

• Cell synchronization: Synchronize cells at different mitotic stages (using nocodazole or thymidine block)

• Co-immunoprecipitation: Use KLHL13 antibodies to pull down the complex and probe for Aurora B

• Immunofluorescence co-localization: Perform dual staining of KLHL13 and Aurora B during mitosis

• Live-cell imaging: Track fluorescently tagged proteins through mitosis

• In vitro ubiquitination assays: Reconstitute the CUL3-KLHL9-KLHL13 complex with recombinant proteins

Research has shown that the BCR(KLHL9-KLHL13) E3 ubiquitin ligase complex mediates Aurora B ubiquitination, controlling its dynamic behavior on mitotic chromosomes and coordinating faithful mitotic progression .

How do you troubleshoot inconsistent results when using KLHL13 antibodies in different cell types?

When encountering inconsistent results across cell types:

• Verify KLHL13 expression levels: Check mRNA expression databases for expected expression in your cell types

• Optimize lysis conditions: Different cell types may require adjusted lysis buffers to efficiently extract KLHL13

• Consider post-translational modifications: KLHL13 may undergo different modifications in various cell types

• Evaluate cell cycle status: KLHL13 levels and localization change during cell cycle progression

• Test multiple antibodies: Use antibodies targeting different epitopes to confirm results

KLHL13 expression varies significantly across tissues, with higher expression in brain and testis, which may explain detection variability in different cell types .

How can KLHL13 antibodies be used to study its role in insulin resistance and metabolic disorders?

For investigating KLHL13's role in insulin resistance:

• Tissue-specific analysis: Compare KLHL13 expression in adipose tissue from normal vs. insulin-resistant subjects

• Co-immunoprecipitation: Use KLHL13 antibodies to pull down complexes and analyze interaction with IRS1

• siRNA knockdown experiments: Assess effects of KLHL13 depletion on insulin signaling

• High-fat diet models: Monitor KLHL13 expression changes in response to diet-induced obesity

Research indicates that KLHL13, along with KLHL9, forms a CUL3-based E3 ubiquitin ligase complex that promotes proteasomal degradation of IRS1, potentially contributing to insulin resistance . Adipose tissue KLHL13 mRNA expression positively correlates with body mass index in humans, suggesting a link to obesity .

Table 1: BioID analysis showing interaction between KLHL13 and IRS1 .

What is the current understanding of KLHL13's role in cancer, and how can antibodies help elucidate these mechanisms?

To investigate KLHL13's role in cancer:

• Expression analysis: Compare KLHL13 levels in tumor vs. normal tissue using IHC

• Substrate identification: Use immunoprecipitation to identify cancer-relevant substrates

• Cell cycle regulation: Evaluate KLHL13's impact on mitotic progression in cancer cells

• Correlation studies: Analyze relationships between KLHL13 expression and patient outcomes

• Functional studies: Assess effects of KLHL13 knockdown/overexpression on cancer cell proliferation

While specific KLHL13 roles in cancer have not been extensively characterized, the broader KLHL family has been implicated in tumorigenesis through ubiquitination of target substrates . For example, KLHL38 promotes lung cancer by targeting PTEN for degradation, whereas KLHL18 acts as a tumor suppressor by inhibiting the PI3K/AKT pathway .

How can researchers investigate KLHL13's potential role in T cell regulation and immunotherapy?

To explore KLHL13's role in T cell regulation:

• Expression analysis: Compare KLHL13 levels in different T cell subsets and activation states

• Co-immunoprecipitation: Investigate potential interactions with immune checkpoint proteins

• T cell functional assays: Assess impact of KLHL13 knockdown on T cell activation and cytokine production

• Patient sample analysis: Correlate KLHL13 expression with response to immunotherapy

Research has shown that while KLHL13 appears in mass spectrometry analyses of immune checkpoints, it does not directly interact with PD-1, unlike its family member KLHL22 . KLHL22 maintains PD-1 homeostasis and prevents excessive T cell suppression, suggesting distinct roles for different KLHL family members in immune regulation.

How do monoclonal and polyclonal KLHL13 antibodies compare in research applications?

Comparison of antibody types for KLHL13 detection:

When choosing between antibody types, researchers should consider their specific application needs and the importance of reproducibility versus sensitivity in their experimental design.

What are the latest methodological advances in studying KLHL13-mediated protein ubiquitination?

Recent methodological advances include:

• Proximity-dependent biotin identification (BioID): This technique has successfully identified IRS1 as a KLHL13 interactor, revealing its role in insulin signaling

• CRISPR-Cas9 knockout validation: Generation of isogenic cell lines enables rigorous antibody validation and substrate identification

• Ubiquitin remnant profiling: Mass spectrometry-based identification of ubiquitination sites on KLHL13 substrates

• Reconstituted in vitro ubiquitination systems: Allow mechanistic studies of KLHL13-CUL3 complex activity

• Live-cell imaging of ubiquitination dynamics: Enables real-time monitoring of KLHL13-mediated substrate degradation

These advanced techniques provide researchers with powerful tools to characterize KLHL13's role in the ubiquitin-proteasome system with unprecedented resolution.

How can researchers integrate multi-omics approaches with KLHL13 antibody-based techniques?

Integrating multi-omics with antibody-based techniques:

• Combine RNA-seq with immunoblotting: Correlate KLHL13 mRNA and protein levels across conditions

• Integrate proteomics with immunoprecipitation: Identify novel KLHL13 interactors and validate by co-IP

• Phospho-proteomics with immunofluorescence: Link phosphorylation events to KLHL13 localization

• ChIP-seq with transcription factor immunoprecipitation: Investigate transcriptional regulation of KLHL13

• Clinical correlations: Connect KLHL13 expression patterns with clinical outcomes and molecular subtypes