KLHL29 Antibody

What is KLHL29 and what structural domains characterize it?

KLHL29 (Kelch-like protein 29) is a human protein that functions as part of the Cul3-RING ubiquitin ligase complex, suggesting involvement in protein ubiquitination pathways. Structurally, KLHL29 contains one BTB (POZ) domain and six Kelch repeats. The BTB domain typically mediates protein-protein interactions, particularly with Cullin-3, while the Kelch repeats form a β-propeller structure that likely facilitates substrate recognition. The protein is also known by alternative names including KBTBD9 and KIAA1921 . The complete sequence is not known with absolute certainty, but current annotations are based on homology with the mouse sequence ortholog . The observed molecular weight is approximately 72 kDa, while the calculated molecular weight is 94228 Da, suggesting potential post-translational modifications or processing .

What tissue expression patterns have been documented for KLHL29?

KLHL29 exhibits a relatively specific tissue expression pattern based on immunohistochemical analyses. According to antibody validation data, KLHL29 protein expression has been detected in several tissues including:

This expression pattern indicates potential roles in neural function, immune regulation, and reproductive biology . For researchers planning to study KLHL29 in specific contexts, selecting appropriate tissue models based on this expression profile is recommended for optimal detection and functional characterization.

What applications are KLHL29 antibodies validated for?

KLHL29 antibodies have been validated for multiple experimental applications, each requiring specific optimization parameters:

It's important to note that each antibody may perform differently across applications despite targeting the same protein. Researchers should validate each antibody in their specific experimental system before proceeding with critical experiments, as performance can vary based on sample preparation, detection methods, and experimental conditions .

How should KLHL29 antibodies be properly stored and handled for optimal performance?

Proper storage and handling of KLHL29 antibodies are critical for maintaining their specificity and activity over time. Based on manufacturer recommendations:

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

• Short-term/frequent use: Store at 4°C for up to one month

• Storage buffer: Most KLHL29 antibodies are supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Freeze-thaw cycles: Minimize repeated freeze-thaw cycles, which can cause protein denaturation and reduced antibody performance

• Shipping conditions: Typically shipped on wet ice

• Working aliquots: For frequent use, prepare small working aliquots to avoid repeated freezing and thawing of the entire stock

Following these guidelines ensures maximum antibody performance and extends the useful life of these valuable research reagents.

How can I validate the specificity of KLHL29 antibodies in my experimental system?

Rigorous validation of KLHL29 antibody specificity is essential for generating reliable research data. A comprehensive validation strategy should include:

Genetic Validation Approaches:

• KLHL29 knockout/knockdown models as negative controls

• KLHL29 overexpression systems as positive controls

• Comparison of signal patterns between wildtype and genetically modified samples

Biochemical Validation Methods:

• Peptide competition assays using the immunizing peptide (e.g., peptides derived from amino acids 261-310 for some KLHL29 antibodies)

• Western blot analysis confirming detection at the expected molecular weight (~72 kDa observed)

• Multiple antibody approach using different KLHL29 antibodies targeting distinct epitopes

Cross-Species Validation:

• Testing antibody reactivity across species when appropriate (some KLHL29 antibodies are validated for both human and mouse)

• Considering epitope conservation across species when interpreting results

Application-Specific Validation:

• For immunohistochemistry, include tissue types known to express or lack KLHL29

• For immunofluorescence, include subcellular localization controls reflecting KLHL29's role in the Cul3-RING complex

• For protein interaction studies, include appropriate negative controls and reciprocal co-immunoprecipitations

These validation steps should be documented and included in publications to strengthen the reliability of findings related to KLHL29 function and expression.

What are the optimal conditions for detecting endogenous versus overexpressed KLHL29?

Detecting endogenous KLHL29 versus overexpressed protein presents distinct methodological challenges requiring different optimization strategies:

Endogenous KLHL29 Detection:

• Use highly sensitive detection methods such as enhanced chemiluminescence for Western blots

• Optimize antibody concentration and incubation time (typically higher concentrations or longer incubations than for overexpressed protein)

• Consider sample enrichment through subcellular fractionation focusing on the Cul3-RING complex components

• Select tissues or cell lines with documented KLHL29 expression (brain, melanoma, placenta, spleen)

• Include appropriate positive controls (tissues known to express KLHL29) and negative controls (KLHL29 knockout samples)

Overexpressed KLHL29 Detection:

• Use inducible expression systems to achieve controlled expression levels

• Consider potential artifacts from non-physiological expression levels

• Be aware of potential mislocalization or altered interaction profiles

• For tagged constructs, verify that tags don't interfere with antibody epitopes

• Compare multiple detection methods (antibody detection vs. tag detection)

Quantitative Comparison:

• When comparing endogenous and overexpressed KLHL29, ensure detection is within the linear range

• Use appropriate normalization methods (housekeeping proteins or total protein stains)

• Consider that overexpression may alter the protein's stability, modification state, or localization

This methodological approach ensures reliable detection across different experimental systems while minimizing artifacts.

How does KLHL29's role in the Cul3-RING ubiquitin ligase complex affect experimental design?

KLHL29's function in the Cul3-RING ubiquitin ligase complex has significant implications for experimental design and interpretation:

Sample Preparation Considerations:

• Include proteasome inhibitors (e.g., MG132) when studying potential KLHL29 substrates

• Use lysis conditions that preserve protein-protein interactions (non-denaturing buffers)

• Consider studying ubiquitination dynamics with ubiquitin immunoprecipitation assays

• Include deubiquitinating enzyme inhibitors to preserve ubiquitin modifications

Experimental Design Elements:

• Design time-course experiments to capture potentially transient ubiquitination events

• Consider studying KLHL29 function in synchronized cells to detect cell-cycle dependencies

• Include other Cul3-RING ligase components in functional studies

• Compare wild-type KLHL29 with domain mutants affecting BTB or Kelch repeats

Potential Substrate Identification:

• Combine KLHL29 overexpression/depletion with proteomics approaches

• Look for proteins whose stability changes with KLHL29 manipulation

• Consider proximity labeling approaches (BioID, APEX) to identify interaction partners

Interaction Verification:

• Use co-immunoprecipitation assays with antibodies against both KLHL29 and known Cul3-RING components

• Consider proximity ligation assays to visualize interactions in situ

• Verify interactions with purified components in vitro

Understanding KLHL29's molecular context allows for more precise experimental designs and more accurate interpretation of results related to its biological function.

How can next-generation sequencing approaches enhance KLHL29 antibody research?

Next-generation sequencing (NGS) technologies provide powerful complementary approaches to antibody-based KLHL29 research:

Transcriptomic Analysis:

• Use RNA-seq to correlate KLHL29 mRNA expression with protein levels detected by antibodies

• Identify potential KLHL29 isoforms through transcript analysis

• Study co-expression patterns to predict functional relationships

• Identify cell types or conditions with high KLHL29 expression for focused antibody studies

Immune Repertoire Analysis:

• For researchers developing new KLHL29 antibodies, NGS of antibody repertoires can enhance discovery processes

• Long-read NGS enables paired VH/VL analysis for more effective antibody development

• NGS can facilitate affinity maturation studies when developing high-affinity KLHL29 antibodies

Functional Genomics Integration:

• Combine CRISPR screens with KLHL29 antibody studies to identify functional pathways

• Correlate genetic perturbations with changes in KLHL29 protein levels or modifications

• Use ChIP-seq to study potential transcriptional regulation of KLHL29 expression

Methodological Considerations:

• When integrating NGS data with antibody-based studies, consider that mRNA and protein levels may not always correlate

• Use appropriate statistical methods for integrating diverse data types

• Validate key findings from NGS approaches with orthogonal antibody-based methods

This integration of NGS with traditional antibody-based approaches provides a more comprehensive understanding of KLHL29 biology and function .

What are the considerations for using KLHL29 antibodies in co-localization studies?

Co-localization studies to investigate KLHL29's interactions and subcellular distribution require careful methodological planning:

Antibody Compatibility:

• Ensure primary antibodies are raised in different host species (most KLHL29 antibodies are rabbit-derived)

• If using multiple rabbit antibodies, consider direct conjugation or sequential staining protocols

• Validate each antibody individually before attempting co-staining experiments

Technical Optimization:

• Test multiple fixation methods (paraformaldehyde, methanol, acetone) to preserve both KLHL29 and co-staining targets

• Optimize permeabilization conditions to ensure antibody access while preserving structural integrity

• Consider detergent selection based on KLHL29's subcellular localization (Cul3-RING complex)

Resolution Considerations:

• Standard confocal microscopy has ~200nm resolution limit

• For definitive co-localization, consider super-resolution techniques (STED, STORM, PALM)

• Proximity ligation assay (PLA) can confirm close proximity (<40nm) between KLHL29 and potential interactors

Quantitative Analysis:

• Use appropriate co-localization coefficients (Pearson's, Manders', etc.)

• Establish thresholds based on control stainings

• Consider 3D analysis rather than single optical sections

• Use specialized co-localization software for unbiased analysis

Validation Controls:

• Include single-stained controls to check for bleed-through

• Use biologically relevant negative controls (proteins known not to interact with KLHL29)

• Include positive controls (other components of the Cul3-RING complex)

• Validate key findings with biochemical interaction assays

These methodological considerations ensure reliable co-localization data that accurately reflects KLHL29's biological context.

How can I troubleshoot non-specific binding when using KLHL29 antibodies?

Non-specific binding is a common challenge when working with antibodies, including those against KLHL29. A systematic troubleshooting approach includes:

Antibody Dilution Optimization:

• Create a dilution series starting with manufacturer recommendations (e.g., 1:100-1:300 for IHC)

• Test both more concentrated and more dilute conditions than recommended

• Identify the optimal concentration that maximizes specific signal while minimizing background

• Consider that different applications may require different optimal dilutions

Blocking Protocol Enhancement:

• Test alternative blocking agents (BSA, normal serum from secondary antibody host, commercial blockers)

• Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

• For cells/tissues, consider adding 0.1-0.3% Triton X-100 to the blocking solution

• For Western blots, test different blockers such as 5% milk or commercial blocking reagents

Washing Optimization:

• Increase number and duration of washing steps

• Use gentle agitation during washes to improve efficiency

• Consider adding low concentrations of detergent (0.05-0.1% Tween-20) to wash buffer

• For immunohistochemistry/immunofluorescence, ensure complete removal of wash buffer between steps

Sample Preparation Refinement:

• Test different fixation methods for immunohistochemistry/immunofluorescence

• Optimize protein loading for Western blots to prevent oversaturation

• Consider native versus denaturing conditions based on the epitope recognized

• Test freshly prepared versus stored samples to assess stability issues

Controls Implementation:

• Include a no-primary antibody control to assess secondary antibody specificity

• Use KLHL29 knockdown/knockout samples as negative controls

• Perform peptide competition assays with the immunizing peptide

• Compare results with alternative KLHL29 antibodies targeting different epitopes

This systematic approach will help identify the source of non-specific binding and lead to optimized protocols for specific KLHL29 detection.

What controls are essential when studying KLHL29 in knockout or knockdown experiments?

Genetic Manipulation Controls:

• Use multiple independent siRNAs/shRNAs targeting different KLHL29 regions to control for off-target effects

• For CRISPR-Cas9 knockout, design multiple guide RNAs and verify editing by sequencing

• Include non-targeting controls (scrambled siRNA, non-targeting gRNA)

• Quantify knockdown/knockout efficiency at both mRNA and protein levels using validated KLHL29 antibodies

Rescue Experiments:

• Re-express KLHL29 in knockout cells to confirm phenotype specificity

• Use expression constructs resistant to siRNA/shRNA for rescue in knockdown experiments

• Consider domain-specific rescue to map functional requirements

• Ensure rescue constructs express at physiological levels

Functional Controls:

• Assess expression of related Kelch-like family proteins that might compensate for KLHL29 loss

• Monitor the integrity of the Cul3-RING complex in the absence of KLHL29

• Examine effects on known ubiquitination pathways as functional readouts

• Include positive controls for phenotypic assays

Validation Across Methods:

• Confirm key findings using orthogonal approaches

• Combine genetic approaches with pharmacological interventions when possible

• Validate in multiple cell types where KLHL29 is expressed (brain, melanoma, placenta, spleen cells)

This comprehensive control strategy ensures that observed phenotypes are specifically attributed to KLHL29 function rather than experimental artifacts.

How do different epitopes in KLHL29 antibodies affect detection of protein variants?

The epitope specificity of KLHL29 antibodies has important implications for detecting different protein forms:

Epitope Mapping Considerations:

• Commercial KLHL29 antibodies target different regions, including amino acids 261-310 in some products

• Other antibodies target alternative regions, such as the immunogen sequence used by Sigma-Aldrich (amino acids from regions containing BTB domain or Kelch repeats)

• These different epitopes may exhibit differential accessibility depending on protein conformation

Detection of Protein Variants:

• Antibodies targeting different domains may detect distinct subsets of KLHL29 variants

• N-terminal epitope antibodies may detect truncated C-terminal variants but miss N-terminal truncations

• C-terminal epitope antibodies show the opposite pattern

• Consider using multiple antibodies targeting different regions for comprehensive detection

Post-translational Modification Effects:

• Epitopes containing modification sites may show differential detection depending on modification status

• The difference between observed (72 kDa) and calculated (94 kDa) molecular weights suggests potential modifications

• Consider phosphorylation, ubiquitination, or other modifications that might affect epitope recognition

• Test detection under conditions that alter modification status (phosphatase treatment, deubiquitinase treatment)

Experimental Validation Approaches:

• Compare detection patterns between multiple KLHL29 antibodies in the same samples

• Express defined KLHL29 variants and test detection with different antibodies

• Use domain deletion constructs to map epitope regions precisely

• Verify critical findings with mass spectrometry or other antibody-independent methods

Understanding these epitope-specific effects is crucial for accurate interpretation of KLHL29 expression and function studies.

How can therapeutic antibody gene transfer approaches be applied to KLHL29 research?

Gene transfer approaches for antibody expression represent an emerging technology with potential applications in KLHL29 research:

Methodological Approaches:

• DNA-based delivery of anti-KLHL29 antibody genes using electroporation or viral vectors

• Development of inducible expression systems for temporal control of anti-KLHL29 antibodies

• Targeting antibody expression to specific tissues where KLHL29 functions

• Integration with CRISPR-based gene editing for combined genetic manipulation and antibody expression

Research Applications:

• Targeted inhibition of KLHL29 function in specific tissues or cell types

• Long-term expression of anti-KLHL29 antibodies for chronic studies

• Development of intrabodies targeting specific KLHL29 domains

• Combined knockdown and antibody-based functional blocking approaches

Technical Considerations:

• Selection of appropriate antibody formats (scFv, Fab, full IgG)

• Optimization of codon usage for expression in specific model systems

• Consideration of immunogenicity in in vivo applications

• Control of antibody production levels to avoid toxicity

Challenges:

• Difficulty controlling antibody production following gene transfer

• Potential for autoimmune reactions if antibodies target endogenous KLHL29

• Need for fully human antibody sequences for long-term expression

• Delivery challenges to relevant KLHL29-expressing tissues

While this approach has challenges, it offers unique possibilities for studying KLHL29 function in ways not possible with conventional approaches.

How does V-gene allelic polymorphism affect KLHL29 antibody development and performance?

Recent research has highlighted the importance of immunoglobulin V-gene allelic polymorphisms in antibody function, with implications for KLHL29 antibody development:

Impact on Antibody Development:

• V-gene allelic polymorphisms in antibody paratopes can determine binding activity to antigens like KLHL29

• Researchers developing new KLHL29 antibodies should consider genetic diversity in their immunization strategies

• Minor V-gene allelic polymorphisms, even with low frequency, can significantly impact antibody performance

• Different animal models may produce varying antibody responses due to genetic differences

Performance Considerations:

• Antibodies raised against KLHL29 may show variable performance across different applications due to structural effects of V-gene variations

• When selecting commercial KLHL29 antibodies, researchers should evaluate performance in their specific experimental system

• Batch-to-batch variation in polyclonal antibodies may partly result from V-gene polymorphisms

• Validation across multiple antibody clones becomes more important given this potential variability

Future Directions:

• Next-generation sequencing of antibody repertoires can help identify optimal anti-KLHL29 antibody candidates

• Structural biology approaches can predict how V-gene variations affect interaction with KLHL29 epitopes

• Computational modeling may help design KLHL29 immunogens that generate more consistent antibody responses

• Understanding V-gene polymorphism effects could lead to more reproducible KLHL29 antibody performance

This emerging understanding of genetic influences on antibody function has important implications for both developing and using KLHL29 antibodies in research contexts.

What novel methodologies are emerging for rapid development of KLHL29-targeting antibodies?

Recent technological advances are revolutionizing antibody development with potential applications for KLHL29 research:

Next-Generation Sequencing Integration:

• NGS analysis of antibody repertoires accelerates identification of KLHL29-specific antibodies

• Computational approaches combining NGS with structural modeling reveal sequence-structure relationships

• Long-read NGS technologies enable paired analysis of heavy and light chains for more effective antibody discovery

• Bioinformatic tools can predict affinity and specificity based on sequence characteristics

Novel Expression Vector Systems:

• Golden Gate-based dual-expression vectors enable rapid screening of recombinant monoclonal antibodies

• In-vivo biotinylation during expression facilitates downstream applications

• Improvements in mammalian expression systems increase efficiency of antibody production

• These advances could accelerate development of new KLHL29-specific antibodies

Innovative B-cell Isolation Technologies:

• Direct immortalization of B cells by gene reprogramming using Epstein-Barr virus or retroviral vectors

• Single-cell culture screening methods to identify KLHL29-specific antibody-producing cells

• Advanced fusion partner cell lines like SPYMEG enhance human hybridoma production

• These approaches offer advantages over traditional hybridoma technology

Computational and Structural Approaches:

• Antibody modeling leveraging structural data to predict KLHL29 binding characteristics

• Machine learning methods to dissect naïve and antigen-driven antibody repertoire convergence

• Structural analysis of antibody affinity maturation to guide KLHL29 antibody optimization

• Integration of computational predictions with experimental validation

These emerging methodologies promise to accelerate the development of high-quality KLHL29 antibodies while reducing development time and resources.