klhl26 Antibody

What is KLHL26 and what structural domains are important for its function?

KLHL26 belongs to the Kelch-like protein family, which plays essential roles in striated muscle development by recruiting specific targets for CUL3-mediated ubiquitination and degradation. The protein contains three key functional domains: the N-terminal BTB (Broad-Complex, Tramtrack, and Bric-a-brac) domain, the central BACK (BTB And C-terminal Kelch) domain, and C-terminal Kelch domains .

Within this structure, the BTB and BACK domains facilitate assembly with CUL3, a component of E3 ubiquitin ligase complexes, while the Kelch β-propeller domain is responsible for target recognition . These domains position KLHL26 as an adaptor protein that recruits specific proteins for ubiquitination and subsequent degradation via the ubiquitin-proteasome system (UPS) .

Recent structural modeling suggests that variants affecting key residues (such as p.R237C in the BACK domain) can disrupt electrostatic interactions when binding to components of the ubiquitin proteasome, specifically altering interactions with CUL3 .

What disease associations have been identified for KLHL26 variants?

A novel missense variant in KLHL26 (c.709C>T p.R237C) has been identified in a multigenerational family with autosomal dominant inheritance of Ebstein's anomaly (EA) with left ventricular noncompaction (LVNC) . EA is a rare congenital heart disease affecting the tricuspid valve and right ventricle, while LVNC is a cardiomyopathy characterized by excessive trabeculations in the left ventricle .

In this family study, the KLHL26 variant segregates exclusively with affected individuals (FBAT p < .05), suggesting a strong genotype-phenotype correlation . Through protein structure prediction modeling, researchers demonstrated that this variant likely disrupts KLHL26's ability to properly interact with CUL3, potentially affecting the ubiquitin-mediated degradation of proteins crucial for cardiac development .

iPSC-derived cardiomyocytes carrying this variant exhibit multiple abnormalities, including:

• Distended endoplasmic reticulum and sarcoplasmic reticulum

• Dysmorphic mitochondria

• Decreased contraction rates

• Altered calcium transients

• Increased cell proliferation

Transcriptomic analyses revealed suppression of the "structural constituent of muscle" pathway and activation of the "ER lumen" pathway in affected cells, providing mechanistic insights into the pathology .

What is the normal expression pattern of KLHL26 in tissues?

KLHL26 shows distinct tissue-specific expression patterns:

• Cardiac tissues: RNA-seq analysis shows KLHL26 expression during cardiomyocyte differentiation, suggesting its importance in heart development .

• Lymphoid tissues: Immunostaining reveals KLHL26 protein expression predominantly in germinal center B cells, with no restriction to either light or dark zones of the germinal center .

• B-cell subpopulations: At the single-cell level, KLHL26 expression varies across germinal center B-cell subpopulations, with highest expression in transitional B cells and downregulation in cells undergoing dark zone and memory B-cell differentiation .

• Subcellular localization: Colocalization studies demonstrate that KLHL26 localizes to perinuclear vesicles concentrated near the Golgi apparatus .

• Vascular system: KLHL26 is highly expressed in arterial cells .

In diffuse large B-cell lymphoma (DLBCL), two distinct expression patterns have been observed: a germinal center-like pattern (KLHL26 GC+) and an aberrant pattern with absent or cytosolic expression (KLHL26 GC-) . The GC+ phenotype is enriched in germinal center B-cell-like (GCB) DLBCL subtypes, while 44% of activated B-cell-like (ABC) DLBCLs show the GC- pattern .

What types of KLHL26 antibodies are commercially available for research?

Several types of KLHL26 antibodies are available for research applications:

Additionally, various CRISPR/Cas9 tools for KLHL26 manipulation are available, including knockout plasmids, HDR plasmids, double nickase plasmids, and CRISPR activation products for both human and mouse KLHL26 .

The antibodies target different regions of the KLHL26 protein, with some specifically targeting the C-terminal region containing the Kelch domains . This diversity allows researchers to select antibodies appropriate for specific experimental needs and applications.

What epitopes are targeted by commonly used KLHL26 antibodies?

Different KLHL26 antibodies target specific epitopes that may affect their utility in various applications:

• C-terminal Region Antibodies:

  • Aviva Systems Biology's KLHL26 Antibody (ARP39346_T100) targets the sequence: "EAGCCLLERKIYIVGGYNWRLNNVTGIVQVYNTDTDEWERDLHFPESFAG"

  • This region includes parts of the Kelch domains responsible for substrate recognition

• Immunogen-Specific Antibodies:

  • Thermo Fisher's KLHL26 Polyclonal Antibody (PA5-54688) uses an immunogen sequence: "LAVEYYVPET DQWTSVSPMR AGQSEAGCCL LERKIYIVGG YNWRLNNVTG IVQVYNTDTD EWERDLHFPE SF"

  • This sequence partially overlaps with the C-terminal region targeted by the Aviva antibody

• Fusion Protein Antibodies:

  • Proteintech's KLHL26 antibody (17216-1-AP) is raised against a fusion protein Ag11002

Understanding the targeted epitope is crucial when:

• Studying variants like p.R237C, as antibodies targeting regions containing the variant might show altered binding

• Investigating domain-specific functions of KLHL26

• Examining protein-protein interactions that might mask certain epitopes

• Detecting post-translational modifications that could affect antibody binding

How should researchers validate KLHL26 antibodies for specific applications?

Rigorous validation is essential for obtaining reliable results with KLHL26 antibodies. A comprehensive validation strategy includes:

• Specificity Testing:

  • Positive controls: Use tissues or cell lines with known KLHL26 expression, such as germinal center B cells, Jurkat cells (confirmed to express KLHL26 by BioGPS gene expression data ), or cardiomyocyte models.

  • Negative controls: Generate KLHL26 knockdown/knockout models using available CRISPR/Cas9 tools .

  • Absorption tests: Pre-incubate the antibody with its immunizing peptide to confirm binding specificity.

• Application-Specific Validation:

  For Western Blotting:

  • Verify the molecular weight (calculated molecular weight: 68 kDa)

  • Confirm band reduction/absence in KLHL26 knockdown/knockout samples

  • Test reproducibility across different sample preparations

  For Immunohistochemistry:

  • Compare staining patterns with known KLHL26 localization (perinuclear vesicles near Golgi)

  • Use positive control tissues (lymphoid tissues with germinal centers, human stomach )

  • Include appropriate negative controls (primary antibody omission, non-expressing tissues)

  For Immunofluorescence:

  • Verify subcellular localization consistent with published findings

  • Perform colocalization studies with Golgi markers

  • Include specificity controls (blocking peptides, knockdown samples)

• Cross-Validation:

  • Compare results from multiple antibodies targeting different KLHL26 epitopes

  • Correlate protein detection with mRNA expression using qPCR

  • Validate across multiple experimental techniques

• Correlation with Disease Models:

  • Test antibody performance in models of KLHL26-associated conditions (EA/LVNC)

  • Assess ability to distinguish between wild-type and variant (p.R237C) KLHL26 if relevant

  • Verify expected expression patterns in disease tissues

What are the optimal protocols for detecting KLHL26 in Western blotting?

Based on available resources and general principles for Western blotting, the following protocol is recommended for KLHL26 detection:

Sample Preparation:

• Prepare whole cell lysates from tissues or cells expressing KLHL26

• For optimal extraction of membrane-associated proteins like KLHL26, use lysis buffers containing appropriate detergents

• Include protease inhibitors to prevent degradation

• Use 10% acrylamide gels for effective separation of KLHL26 (68 kDa)

Electrophoresis and Transfer:

• Load positive controls (e.g., Jurkat cell lysate)

• Include negative controls (KLHL26 knockdown/knockout samples if available)

• Transfer proteins to PVDF or nitrocellulose membranes using standard conditions

Antibody Incubation:

• Block membranes with 5% non-fat dry milk or BSA in TBST

• For Atlas Antibodies HPA023074 or Aviva Systems Biology ARP39346_T100, follow the manufacturer's recommended dilution for Western blotting

• Incubate with primary antibody overnight at 4°C

• Wash thoroughly with TBST (3-5 washes, 5-10 minutes each)

• Incubate with appropriate HRP-conjugated secondary antibody

Detection and Validation:

• Use ECL or other detection systems appropriate for the expected expression level

• Verify the expected molecular weight (68 kDa)

• For validation, consider stripping and reprobing with another KLHL26 antibody targeting a different epitope

• Compare with KLHL26 mRNA expression using qPCR methods as described for KLHL26 transcript analysis

Optimization Considerations:

• If background is high, increase washing duration/frequency

• If signal is weak, consider longer exposure times or more sensitive detection methods

• For tissues with low KLHL26 expression, consider enrichment methods before Western blotting

How can researchers study KLHL26 in immunohistochemistry applications?

Several KLHL26 antibodies have been validated for immunohistochemistry, including Thermo Fisher's PA5-54688 and Atlas Antibodies' HPA023074. The following protocol is recommended:

Tissue Preparation:

• Use appropriate positive control tissues (lymphoid tissues with germinal centers, human stomach)

• Standard formalin fixation and paraffin embedding (FFPE) is suitable based on validated antibodies

• Section tissues at 4-5 μm thickness

Antigen Retrieval:

• Perform heat-induced epitope retrieval (HIER)

• Test both citrate buffer (pH 6.0) and EDTA buffer (pH 9.0) to determine optimal conditions

• Use pressure cooker or microwave methods for consistent retrieval

Staining Protocol:

• Block endogenous peroxidase activity with hydrogen peroxide

• Block non-specific binding with appropriate serum or commercial blocking solutions

• Incubate with primary antibody at manufacturer's recommended dilution

• Use appropriate detection systems (polymer-based or ABC-based)

• Develop with DAB and counterstain with hematoxylin

Expected Staining Pattern:
Based on published research, KLHL26 shows the following distribution:

• In normal lymphoid tissues: Mainly in germinal center B cells

• Subcellular localization: Perinuclear vesicles concentrated near the Golgi apparatus

• In DLBCL samples: Two distinct patterns - KLHL26 GC+ (germinal center-like expression) and KLHL26 GC- (absent or cytosolic expression)

Controls and Validation:

• Include positive controls (tissues known to express KLHL26)

• Include negative controls (primary antibody omission)

• For further validation, compare with immunofluorescence studies

• Consider double staining with markers of germinal centers (e.g., BCL6) to confirm the expected co-expression pattern (90% of KLHL26 GC+ samples were also BCL6+ by IHC)

What approaches are effective for studying KLHL26 protein-protein interactions?

Since KLHL26 functions as an adaptor protein in E3 ubiquitin ligase complexes, studying its protein-protein interactions is crucial. The following methodologies are recommended:

Co-Immunoprecipitation (Co-IP):

• Santa Cruz Biotechnology's KLHL26 Antibody (F-7, sc-514015) is validated for immunoprecipitation

• Use mild lysis buffers to preserve protein-protein interactions

• Include appropriate controls (IgG control, input samples)

• Based on structural predictions, focus on interactions with CUL3 and potential substrate proteins

• Compare co-IP results between wild-type KLHL26 and the p.R237C variant to assess the impact on protein interactions

Affinity Purification-Mass Spectrometry (AP-MS):

• Similar approaches to those used for related proteins like KLHL6 can be adapted for KLHL26

• Immunoprecipitate KLHL26 using validated antibodies

• Analyze co-precipitated proteins by mass spectrometry

• Compare interaction profiles under different conditions or between wild-type and variant KLHL26

Proximity Ligation Assay (PLA):

• This technique can visualize protein-protein interactions in situ

• Use antibodies against KLHL26 and its potential interacting partners from different host species

• Apply PLA probes and detection reagents according to standard protocols

• Analyze by fluorescence microscopy to detect and quantify specific interactions

Functional Validation:

• Manipulate expression of potential interaction partners and assess effects on KLHL26 function

• For studying the p.R237C variant specifically, compare its interaction profile with wild-type KLHL26

• Focus on CUL3 interaction, which is predicted to be affected by this variant

• Assess downstream effects on ubiquitination of potential substrate proteins

How can researchers investigate KLHL26's role in the ubiquitin-proteasome system?

KLHL26 is highly likely to function as a substrate adaptor in CUL3-based E3 ubiquitin ligase complexes. The following strategies can help elucidate its role in the ubiquitin-proteasome system:

Identification of KLHL26 Substrates:

• Global Proteomics Approach:

  • Compare protein abundance in wild-type vs. KLHL26 knockout/knockdown cells using mass spectrometry

  • Proteins that accumulate in KLHL26-deficient cells are potential substrates

  • Validate candidates by assessing their half-life in the presence/absence of KLHL26

• Ubiquitinome Analysis:

  • Use ubiquitin remnant profiling (K-ε-GG) mass spectrometry

  • Compare ubiquitinated proteins in control vs. KLHL26-deficient cells

  • Focus on proteins showing decreased ubiquitination when KLHL26 is absent

Characterization of KLHL26-CUL3 E3 Ligase Complex:

• Biochemical Complex Assembly:

  • Use purified components to reconstruct the KLHL26-CUL3-RBX1 complex in vitro

  • Assess complex formation using antibodies against each component

  • Compare wild-type KLHL26 with the p.R237C variant

• Structural Studies:

  • Extend existing protein prediction models that suggest the KLHL26 variant disrupts electrostatic interactions when binding to CUL3

  • Use antibodies to validate structural predictions in cellular contexts