KBTBD2 Antibody

What is KBTBD2 and what cellular functions does it perform?

KBTBD2 (kelch repeat and BTB domain containing 2) is a 623 amino acid protein with a molecular weight of 71.3 kDa that functions as a substrate-specific adapter of the BCR (BTB-CUL3-RBX1) E3 ubiquitin ligase complex. It plays a critical role in regulating the insulin signaling pathway by modulating insulin sensitivity through limiting PIK3R1/p85alpha abundance in adipocytes . The protein is involved in various cellular processes including protein ubiquitination and metabolic regulation, facilitating the ubiquitination and subsequent proteasomal degradation of specific substrate proteins . This activity is crucial for maintaining cellular protein homeostasis and regulating metabolic pathways, with alterations in KBTBD2 function or expression being associated with metabolic disorders such as obesity and type 2 diabetes .

What types of KBTBD2 antibodies are commercially available for research applications?

Multiple types of KBTBD2 antibodies are available for research, including:

Commercially available options include mouse monoclonal antibodies that recognize human, mouse, and rat antigens with applications in Western Blot, Immunofluorescence, Immunocytochemistry, and ELISA . Rabbit polyclonal antibodies conjugated to biotin are also available, generated against specific regions of the human KBTBD2 protein . The choice between these options depends on the specific experimental requirements and target species.

What are the common alternative names and identifiers for KBTBD2?

When searching literature or databases for KBTBD2, researchers should be aware of these alternative identifiers:

• Protein aliases: BTB and kelch domain containing 1, BTB and kelch domain-containing protein 1, kelch repeat and BTB (POZ) domain containing 2, BKLHD1

• Gene aliases: BKLHD1, CGI-73, KIAA1489

• Database identifiers: UniProt ID (Human): Q8IY47, Entrez Gene ID (Human): 25948

Understanding these alternative names is essential when conducting literature searches or when ordering antibodies from different suppliers who may use varied nomenclature.

Which applications are KBTBD2 antibodies validated for, and what are the recommended dilutions?

KBTBD2 antibodies have been validated for multiple applications with specific recommended dilutions:

When performing Western Blot analysis, researchers should expect to detect a band at approximately 60-70 kDa , though the canonical protein has a reported mass of 71.3 kDa . Positive results have been specifically demonstrated in COLO 320 and A549 cell lines . Always perform optimization experiments to determine the ideal dilution for your specific experimental conditions, as sample types and detection methods may require adjustments.

What are the optimal storage conditions for KBTBD2 antibodies to maintain activity?

For optimal preservation of antibody activity, KBTBD2 antibodies should generally be stored at -20°C, where they remain stable for one year after shipment . Some antibodies may be stored at -80°C for longer-term preservation . Avoid repeated freeze-thaw cycles, as this can degrade antibody quality and reduce specificity and sensitivity. Aliquoting antibodies upon receipt is recommended to minimize freeze-thaw cycles. For working dilutions, storage at 4°C for short periods (typically 1-2 weeks) is acceptable, but long-term storage of diluted antibodies is not recommended. Always refer to manufacturer-specific storage recommendations, as formulation buffers may differ.

What controls should be included when using KBTBD2 antibodies in experiments?

Proper controls are essential for validating KBTBD2 antibody specificity and ensuring experimental rigor:

• Positive controls: Use cell lines with confirmed KBTBD2 expression such as COLO 320 or A549 cells, which have shown positive results in Western blot analyses .

• Negative controls: Include samples where KBTBD2 is either not expressed or has been knocked down through siRNA/shRNA techniques.

• Loading controls: When performing Western blots, include housekeeping protein detection (β-actin, GAPDH, etc.) to normalize expression levels.

• Secondary antibody-only controls: Omit primary antibody to identify potential non-specific binding of secondary antibodies.

• Peptide competition assays: Pre-incubate antibody with the immunizing peptide to confirm binding specificity.

For advanced validation, consider using KBTBD2 knockout models or CRISPR-Cas9 edited cell lines as gold-standard negative controls to definitively demonstrate antibody specificity.

How can I troubleshoot weak or absent signal when using KBTBD2 antibodies in Western blot?

When facing weak or absent signals in Western blot experiments with KBTBD2 antibodies, consider these methodological adjustments:

• Sample preparation: Ensure complete protein denaturation and use fresh protease inhibitors. KBTBD2 is involved in ubiquitination pathways, so including deubiquitinase inhibitors may prevent degradation.

• Loading amount: Increase protein loading (50-80 μg may be necessary) as KBTBD2 expression can be relatively low in some tissues/cell lines.

• Antibody concentration: Try increasing primary antibody concentration beyond the recommended 1:500-1:1000 dilution , particularly for polyclonal antibodies.

• Incubation conditions: Extend primary antibody incubation to overnight at 4°C to enhance binding sensitivity.

• Detection system: Switch to a more sensitive detection method (e.g., from colorimetric to chemiluminescence or from standard ECL to enhanced ECL).

• Membrane type: PVDF membranes often provide better protein retention than nitrocellulose for some targets.

• Transfer conditions: Optimize transfer time for high molecular weight proteins (~71 kDa for KBTBD2 ).

If the issue persists, verify KBTBD2 expression in your sample through RT-PCR or consider testing another validated antibody, as different epitopes may be more accessible in your experimental system.

What are potential sources of non-specific binding when using KBTBD2 antibodies in immunofluorescence, and how can they be minimized?

Non-specific binding in immunofluorescence experiments can obscure true KBTBD2 localization. Address these common issues:

• Blocking optimization: Extend blocking time (2-3 hours) or test different blocking agents (BSA, normal serum, commercial blockers) to reduce background.

• Antibody concentration: Titrate the antibody carefully; for IF/ICC applications with KBTBD2 antibodies, often lower concentrations than recommended for WB are optimal .

• Fixation method: Compare paraformaldehyde, methanol, and acetone fixation, as epitope accessibility can vary with different fixation methods.

• Permeabilization conditions: Test different detergents (Triton X-100, Tween-20, saponin) at various concentrations, as over-permeabilization can increase non-specific binding.

• Wash stringency: Increase wash duration or detergent concentration in wash buffers to remove weakly bound antibodies.

• Autofluorescence reduction: Include quenching steps (sodium borohydride, ammonium chloride) to reduce cellular autofluorescence.

• Secondary antibody cross-reactivity: Use highly cross-adsorbed secondary antibodies specific to the host species of your primary antibody.

• Validation with peptide competition: Pre-incubate the antibody with immunizing peptide as a control to identify non-specific binding.

Always include appropriate negative controls and, when possible, validate subcellular localization through complementary techniques like subcellular fractionation.

How can KBTBD2 antibodies be effectively used to study its role in insulin signaling pathways?

KBTBD2 functions as a regulator of the insulin signaling pathway by modulating insulin sensitivity through limiting PIK3R1/p85alpha abundance in adipocytes . To effectively study this role:

• Co-immunoprecipitation (Co-IP) approach: Use KBTBD2 antibodies for Co-IP experiments to pull down interacting proteins within the insulin signaling pathway, particularly PIK3R1/p85alpha, to confirm direct interactions. Follow with mass spectrometry to identify novel interaction partners.

• Proximity ligation assay (PLA): Combine KBTBD2 antibodies with antibodies against insulin pathway components to visualize and quantify protein interactions at the single-molecule level in situ.

• Phosphorylation analysis: After insulin stimulation, perform immunoprecipitation with KBTBD2 antibodies followed by phospho-specific Western blotting to investigate how insulin signaling affects KBTBD2 post-translational modifications.

• Ubiquitination assays: Since KBTBD2 is part of an E3 ubiquitin ligase complex , use KBTBD2 antibodies in conjunction with ubiquitin antibodies to investigate ubiquitination of substrates following insulin receptor activation.

• Translocation studies: Use immunofluorescence with KBTBD2 antibodies to track potential subcellular redistribution of KBTBD2 in response to insulin.

• Metabolic phenotype correlation: Correlate KBTBD2 expression levels (via Western blot quantification) with insulin sensitivity metrics in various cell lines or primary cultures from metabolic disease models.

This multifaceted approach can provide comprehensive insights into KBTBD2's functional role in insulin signaling regulation.

What considerations are important when using KBTBD2 antibodies to investigate its role in metabolic disorders?

When investigating KBTBD2's association with metabolic disorders such as obesity and type 2 diabetes :

• Tissue-specific expression analysis: Use validated KBTBD2 antibodies for immunohistochemistry or Western blot to compare expression across metabolically relevant tissues (adipose, liver, muscle, pancreas) in normal versus disease states.

• Patient sample considerations: When analyzing human samples, account for medication effects, particularly insulin sensitizers or weight management drugs, which might alter KBTBD2 expression or post-translational modifications.

• Animal model selection: Different metabolic disorder models may show variable KBTBD2 regulation; confirm antibody cross-reactivity with the species being studied (orthologs reported in mouse, rat, bovine, etc. ).

• Temporal dynamics: Design time-course experiments to determine whether KBTBD2 alterations precede or follow metabolic dysfunction, using consistent antibody lots for comparable quantification.

• Functional correlation: Correlate KBTBD2 protein levels or localization with functional metabolic parameters such as glucose uptake, insulin sensitivity, or lipid metabolism.

• Intervention studies: Examine how therapeutic interventions affect KBTBD2 levels or activity, potentially identifying it as a biomarker for treatment response.

• Post-translational modification analysis: Investigate whether diabetes or obesity affect KBTBD2 phosphorylation, ubiquitination, or other modifications that might alter its function.

Understanding these considerations will enhance the translational relevance of KBTBD2 research in metabolic disease contexts.

How can researchers effectively study KBTBD2's role in protein ubiquitination using available antibodies?

To investigate KBTBD2's function as a substrate adaptor for the Cullin-RING E3 ubiquitin ligase complex :

• In vitro ubiquitination assay: Immunoprecipitate KBTBD2 using specific antibodies to isolate the associated E3 ligase complex. Add purified E1, E2, ubiquitin, ATP, and potential substrate proteins to assess ubiquitination activity in a controlled system.

• Substrate identification: Combine KBTBD2 immunoprecipitation with mass spectrometry under both control and KBTBD2-depleted conditions to identify differentially ubiquitinated proteins, focusing on PIK3R1/p85alpha and other insulin pathway components.

• Ubiquitin chain linkage analysis: Use KBTBD2 antibodies together with linkage-specific ubiquitin antibodies (K48, K63, etc.) to determine the type of ubiquitin chains KBTBD2-containing complexes generate, which indicates whether targets are destined for degradation (K48) or signaling modification (K63).

• Protein stability assays: Monitor substrate half-life through cycloheximide chase experiments in the presence or absence of KBTBD2 (via knockdown/knockout), followed by Western blot detection using KBTBD2 antibodies.

• Structural interaction studies: Combine KBTBD2 domain mapping (using deletion constructs) with co-immunoprecipitation to determine which domains interact with the BCR complex components versus substrates, using domain-specific antibodies when available.

• Regulation of KBTBD2 activity: Investigate post-translational modifications of KBTBD2 itself that might regulate its adaptor function, using phospho-specific or modification-specific antibodies after immunoprecipitation with general KBTBD2 antibodies.

These approaches provide a comprehensive toolkit for dissecting KBTBD2's mechanistic role in protein ubiquitination and targeted degradation.

How should researchers interpret discrepancies in KBTBD2 molecular weight observed in Western blot experiments?

Researchers may observe variations in KBTBD2 molecular weight between the theoretical 71.3 kDa and the 60-70 kDa range reported for some antibodies . Consider these potential explanations when interpreting discrepancies:

• Post-translational modifications: Ubiquitination (given KBTBD2's role in ubiquitin ligase complexes ) may increase apparent molecular weight, while proteolytic processing might decrease it.

• Isoform detection: Different antibodies may recognize specific isoforms or splice variants of KBTBD2, potentially explaining size variations across antibody clones.

• Experimental conditions: SDS-PAGE running conditions, gel percentage, and buffer systems can affect protein migration. Always include molecular weight markers and run known positive controls.

• Antibody specificity: Some antibodies may detect related proteins with similar epitopes in the kelch repeat or BTB domain families. Validate with knockout/knockdown controls.

• Sample preparation: Incomplete denaturation or protein degradation during sample preparation can shift apparent molecular weight.

To resolve these discrepancies, researchers should:

• Compare results using multiple antibodies targeting different KBTBD2 epitopes

• Verify specificity through siRNA knockdown or CRISPR knockout validation

• Use mass spectrometry to confirm protein identity from excised bands

• Consult UniProt and literature for documented post-translational modifications or isoforms

What considerations are important when comparing KBTBD2 expression across different species or tissues?

When comparing KBTBD2 expression across different biological contexts:

• Epitope conservation: Ensure the antibody's epitope is conserved in the species being studied. While KBTBD2 orthologs have been reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken , epitope sequence variation may affect antibody binding affinity.

• Validation in each species: Even for antibodies labeled as cross-reactive, perform validation in each species of interest using positive and negative controls specific to that organism.

• Tissue-specific considerations:

  • Different tissues may express various isoforms requiring specific antibody selection

  • Tissue-specific post-translational modifications might mask epitopes

  • Background autofluorescence varies by tissue type in immunohistochemistry applications

  • Extraction protocols may need optimization for different tissue types

• Quantification methods: When comparing expression levels:

  • Use consistent loading controls appropriate for the tissues being compared

  • Consider absolute quantification using recombinant protein standards

  • Account for antibody affinity differences when using different antibodies

• Developmental and physiological state: KBTBD2 expression may vary with developmental stage, nutritional status, or disease state, particularly given its role in metabolic regulation .

Addressing these considerations ensures that observed differences reflect true biological variation rather than technical artifacts.