klhl41b Antibody

What is KLHL41 and why is it important in research?

KLHL41 (also known as Krp1, SARCOSIN, kelch-like protein 41, or KBTBD10) is a 68 kDa protein consisting of 606 amino acid residues that is primarily expressed in skeletal muscle. It contains BTB (Broad-Complex, Tramtrack, and Bric-a-brac), BACK, and Kelch-repeat domains. KLHL41 plays a crucial role in skeletal muscle development and differentiation, with its subcellular localization primarily in the endoplasmic reticulum and cytoplasm . The importance of KLHL41 in research stems from its role in muscle development and its association with nemaline myopathy, a severe and sometimes fatal congenital muscle disorder characterized by muscle weakness and the presence of rod-like structures (nemaline bodies) in muscle fibers .

What is the difference between KLHL41 and KLHL41b antibodies?

While KLHL41 refers to the human protein, KLHL41b specifically references the zebrafish (Danio rerio) ortholog of this protein. Antibodies against KLHL41b are specifically raised against and tested for reactivity with the zebrafish protein (Uniprot ID: F1QEG2) . This distinction is important for researchers working with animal models, as species-specific antibodies ensure optimal recognition of the target protein in the specific organism being studied. Human KLHL41 antibodies may not cross-react effectively with zebrafish KLHL41b due to potential sequence differences, making species-specific antibodies essential for accurate experimental results.

What experimental applications are KLHL41b antibodies suitable for?

KLHL41b antibodies have been validated for several key applications:

For optimal results, researchers should perform antibody titration experiments to determine the ideal concentration for their specific experimental conditions. The antibodies are typically provided in a liquid form with preservatives such as sodium azide or Proclin 300 in buffer solutions like PBS with glycerol .

How should I design experiments to study KLHL41's role in ubiquitination processes?

When designing experiments to study KLHL41's role in ubiquitination:

• Co-immunoprecipitation assays: Express KLHL41-FLAG and your protein of interest with a different tag (e.g., V5) in a muscle cell line such as C2C12 myoblasts. Immunoprecipitate using an antibody against your tag of interest and perform western blot analysis to detect KLHL41 interaction .

• Ubiquitination analysis: Co-express KLHL41-FLAG with your protein of interest and HA-tagged ubiquitin. Vary the concentrations of KLHL41 plasmid (0-1.0 ng) while keeping the protein of interest constant (e.g., 2.0 ng). After 48 hours, immunoprecipitate your protein and analyze ubiquitination patterns by western blot using anti-HA antibodies .

• Domain-specific interactions: Create deletion mutants lacking specific domains (BTB, BACK, or Kelch repeats) to determine which domains are essential for specific protein-protein interactions. The BTB domain is particularly important for KLHL41 homodimerization and interactions with CUL3 .

• Ubiquitin mutant analysis: Use HA-tagged ubiquitin mutants (K6R, K11R, K27R, K29R, K33R, K48R, K63R) to identify which type of ubiquitination regulates KLHL41 function. K48R mutation has been shown to reduce KLHL41's ability to stabilize nebulin fragments, suggesting K48-linked polyubiquitination is important for this function .

What are the methodological considerations for investigating KLHL41's role as a molecular chaperone?

To investigate KLHL41's chaperone function:

• Protein aggregation assays: Express your protein of interest (e.g., nebulin fragment) alone or with KLHL41 in cell lines. Extract both soluble and insoluble protein fractions using different detergent concentrations. Compare the distribution of your protein between soluble and insoluble fractions with and without KLHL41 co-expression .

• Immunofluorescence visualization: Express fluorescently tagged versions of your protein of interest with and without KLHL41 to visualize aggregation patterns. Without KLHL41, proteins like nebulin fragments tend to form aggregates, often in the nucleus, while co-expression with KLHL41 typically results in homogeneous cytosolic staining .

• Stability assays: Treat cells with cycloheximide to inhibit protein synthesis and monitor degradation rates of your protein of interest with and without KLHL41 co-expression over time. This approach can reveal how KLHL41 affects protein half-life .

• Proteasome inhibition: Use proteasome inhibitors (e.g., MG132) to determine if KLHL41's stabilizing effect involves preventing proteasomal degradation of target proteins .

Remember that KLHL41's chaperone function appears to be partner-specific, as it has been shown to stabilize nebulin but not necessarily other interaction partners like NRAP or FLNC .

How can I differentiate between the roles of KLHL41 and KLHL40 in experimental models?

Differentiating between KLHL41 and KLHL40 functions requires careful experimental design:

• Binding partner analysis: While both proteins share some common interacting partners (like nebulin), they also have distinct ones. KLHL41 interacts with nebulin and FLNC, whereas KLHL40 interacts with nebulin and LMOD3 .

• Knockdown/knockout models: Generate separate KLHL41 and KLHL40 knockout models and compare phenotypic differences. KLHL41 knockout mice display severe sarcomere disarray, accumulation of nemaline bodies, and perinatal death, similar to human KLHL41-related nemaline myopathy .

• Hierarchical relationship analysis: Express one protein in the absence of the other to determine if they function in a hierarchical manner or independently. For example, in the presence of both NRAP and NEB, KLHL40 preferentially co-immunoprecipitates with NRAP, suggesting differential affinities for shared binding partners .

• Protein rescue experiments: In KLHL41-deficient models, determine if KLHL40 overexpression can rescue the phenotype and vice versa. Partial rescue would suggest overlapping but distinct functions .

• Complex formation analysis: Investigate if KLHL41 and KLHL40 form heterodimers or heteropolymers through co-immunoprecipitation and size exclusion chromatography. The BTB domain of KLHL40 has been shown to be critical for its association with KLHL41 .

What methods should I use to validate KLHL41b antibody specificity in zebrafish models?

For zebrafish KLHL41b antibody validation:

• Western blot with recombinant protein: Run purified recombinant KLHL41b protein alongside zebrafish tissue lysates to confirm the antibody recognizes the correct molecular weight band.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before applying to western blot or immunostaining. Signal elimination confirms specificity.

• Morpholino knockdown: Use morpholinos to knock down klhl41b expression in zebrafish embryos, then compare antibody signal between knockdown and control embryos. Reduced signal in knockdown samples confirms specificity .

• CRISPR/Cas9 knockout: Generate CRISPR-based klhl41b knockout zebrafish and validate antibody specificity by confirming signal loss in homozygous mutants.

• Cross-reactivity testing: Test the antibody against closely related proteins (like KLHL40) to ensure it doesn't cross-react with other family members.

• Immunohistochemistry with appropriate controls: Perform immunostaining on zebrafish muscle tissues, including negative controls (secondary antibody only) and tissue from klhl41b-deficient animals .

How can KLHL41b antibodies contribute to nemaline myopathy research?

KLHL41b antibodies can significantly advance nemaline myopathy research through:

• Protein localization studies: Immunostaining with KLHL41b antibodies can reveal alterations in protein localization within muscle fibers in disease models. In healthy human biopsies, KLHL41 staining predominates over the I-bands of the sarcomere and at perinuclear regions .

• Protein-protein interaction networks: Immunoprecipitation with KLHL41b antibodies followed by mass spectrometry can identify novel interaction partners that might be disrupted in disease states, providing new therapeutic targets.

• High-throughput screening: KLHL41b antibodies can be used in high-throughput screens to identify compounds that stabilize KLHL41 or enhance its chaperone function, potentially leading to therapeutic approaches for nemaline myopathy.

• Biomarker development: Quantification of KLHL41 protein levels in patient samples using KLHL41 antibodies might serve as a biomarker for disease progression or therapeutic response.

• In vivo imaging: Fluorescently labeled KLHL41b antibodies could potentially be used for in vivo imaging of zebrafish models to monitor protein dynamics in real-time during disease progression .

What are the methodological considerations for using KLHL41b antibodies in protein-protein interaction studies?

When using KLHL41b antibodies for protein-protein interaction studies:

• Homogeneous Time-Resolved Fluorescence (HTRF) assay: For direct protein-protein interaction studies, purify recombinant KLHL41b and potential binding partners, then test interactions at different concentrations (e.g., 1, 10, and 100 nM). Higher delta F% values indicate stronger interactions, as seen with KLHL41-NRAP interactions (delta F%=1390) compared to KLHL41-NEB interactions (delta F%=950) .

• Co-immunoprecipitation optimization: For co-IP, consider:

  • Cell lysis conditions: Use buffers containing 0.5-1% NP-40 or Triton X-100 with protease inhibitors

  • Antibody concentration: Typically 1-5 μg antibody per 500 μg of cell lysate

  • Pre-clearing lysates: Use protein A/G beads before antibody addition to reduce non-specific binding

  • Controls: Include IgG control and input samples

• Tandem Affinity Purification (TAP): For identifying novel interaction partners, use TAP with 3xFLAG-HA-KLHL41b followed by mass spectrometry. This approach has successfully identified interactions between KLHL41 and sarcomeric proteins .

• Yeast two-hybrid screening: As a complementary approach, use KLHL41b as bait to screen for binding partners. This method identifies direct protein-protein interactions independent of post-translational modifications .

• Domain mapping: Create deletion mutants lacking specific domains (BTB, BACK, or Kelch repeats) to map the regions required for specific protein interactions. For example, deletion of the BTB domain abolishes KLHL41 homodimerization and CUL3 association .

What are common technical challenges with KLHL41b antibodies and how can I address them?

Common challenges and solutions include:

• High background in Western blots:

  • Increase blocking time (up to 2 hours)

  • Use different blocking agents (5% milk vs. BSA)

  • Increase washing steps and duration

  • Dilute primary antibody further (1:2000 instead of 1:200)

  • Use freshly prepared buffers

• Weak or no signal:

  • Confirm protein expression in your sample (use positive control)

  • Increase antibody concentration

  • Extend primary antibody incubation (overnight at 4°C)

  • Use more sensitive detection methods (ECL-plus vs. standard ECL)

  • Ensure protein transfer efficiency with Ponceau staining

• Multiple bands in Western blot:

  • Consider post-translational modifications (KLHL41 is poly-ubiquitinated)

  • Verify if bands represent different isoforms (up to 2 isoforms reported)

  • Run appropriate controls to distinguish non-specific binding

  • Use peptide competition assays to confirm specificity

• Inconsistent immunoprecipitation results:

  • Optimize lysis conditions to preserve protein interactions

  • Use crosslinking agents to stabilize transient interactions

  • Consider tag interference with protein interactions

  • Try different antibody orientations (pre-bound to beads vs. direct addition to lysate)

How should I validate a new lot of KLHL41b antibody before use in critical experiments?

For rigorous validation of new antibody lots:

• Western blot comparison:

  • Run both old and new antibody lots side-by-side on the same blot

  • Compare band patterns, intensity, and background

  • Use recombinant KLHL41b protein as a positive control

  • Include samples from wild-type and KLHL41b-deficient tissues

• Immunoprecipitation efficiency:

  • Compare protein pull-down efficiency between lots

  • Verify capture of known interaction partners

  • Quantify the percentage of target protein recovered from input

• Epitope verification:

  • Confirm epitope recognition using peptide competition assays

  • For antibodies against specific regions (N-term vs. C-term), verify regional specificity

• Application-specific validation:

  • For immunohistochemistry: Compare staining patterns and intensity

  • For ELISA: Generate standard curves and compare sensitivity and dynamic range

  • For flow cytometry: Compare population separation and mean fluorescence intensity

• Documentation:

  • Record lot number, validation date, and results

  • Store reference samples of validated lots for future comparisons

What are the emerging research areas involving KLHL41 and how might antibodies facilitate these studies?

Emerging research areas and antibody applications include:

• KLHL41 as a poly-ubiquitination sensor: Recent findings suggest KLHL41 may serve as a poly-ubiquitination sensor, offering a new regulatory layer to muscle sarcomeres. Antibodies specifically recognizing poly-ubiquitinated KLHL41 could help monitor this regulatory mechanism in various physiological and pathological conditions .

• KLHL41-KLHL40 heterodimer complex: Evidence suggests KLHL41 and KLHL40 may form heterodimers or polymers to regulate thin filament proteins. Co-immunoprecipitation with KLHL41b antibodies could help characterize these complexes and their functional significance in muscle development and maintenance .

• Non-proteolytic functions of ubiquitination: Unlike typical ubiquitination that leads to protein degradation, KLHL41 poly-ubiquitination appears to stabilize proteins like nebulin. Antibodies that can distinguish different ubiquitination patterns could help elucidate this unconventional mechanism .

• Therapeutic approaches for nemaline myopathy: As KLHL41 functions as a chaperone preventing protein aggregation, antibody-based screening platforms could identify compounds that enhance this activity as potential therapeutics for nemaline myopathy.

• Developmental regulation: KLHL41's role in muscle development suggests temporal regulation of its expression and activity. Antibodies could track KLHL41 expression patterns during development and differentiation to understand critical windows for therapeutic intervention .

How do recent findings about KLHL41's molecular chaperone function impact experimental design considerations?

The discovery of KLHL41's molecular chaperone function has significant implications for experimental design:

• Solubility assays: When studying KLHL41's impact on interacting proteins, researchers should examine both soluble and insoluble fractions. KLHL41 has been shown to shift proteins like nebulin fragments from insoluble to soluble fractions, preventing aggregation .

• Aggregation visualization: Immunofluorescence microscopy should be employed to visualize protein aggregation patterns with and without KLHL41. Without KLHL41, proteins like nebulin fragments form aggregates predominantly in the nucleus, while KLHL41 co-expression promotes homogeneous cytosolic distribution .

• Ubiquitination analysis: When studying KLHL41's impact on protein stability, researchers should assess not only KLHL41's ability to ubiquitinate substrates but also how KLHL41's own ubiquitination status affects its chaperone function. Ubiquitin mutants, particularly K48R, can be used to disrupt KLHL41's stabilizing activity .

• Domain-specific functions: Experiments should include domain deletion mutants to dissect which regions of KLHL41 are required for specific functions. The BTB domain is crucial for KLHL41 poly-ubiquitination and its ability to stabilize nebulin fragments .

• Partner specificity: When investigating KLHL41's chaperone activity, researchers should recognize that this function appears to be partner-specific. While KLHL41 stabilizes nebulin, it doesn't necessarily affect other interacting proteins like NRAP or FLNC. Experimental designs should include multiple potential partners to determine specificity .