KLHL36 Antibody

What is KLHL36 and what is its structural composition?

KLHL36 is a 616 amino acid protein that contains six Kelch repeats, one BTB/POZ domain, and one BTB/Kelch associated (BACK) domain. These structural elements are critical for protein-protein interactions, particularly for its role in ubiquitination processes. The protein is encoded by the KLHL36 gene (also known as C16orf44 or FLJ12543) and exists in two isoforms resulting from alternative splicing events .

What is the primary function of KLHL36 in cellular processes?

KLHL36 primarily functions as a substrate-specific adapter within E3 ubiquitin-protein ligase complexes. It mediates the transfer of ubiquitin from E2 ubiquitin-conjugating enzymes to target proteins that are destined for degradation. Notably, KLHL36 interacts with CUL-3, a member of the cullin family that participates in the selective targeting of proteins for ubiquitin-mediated proteolysis. This interaction is crucial for cellular processes including cell cycle regulation and signal transduction .

What types of KLHL36 antibodies are available for research purposes?

Several types of KLHL36 antibodies are available for research:

• Host species: Mouse and rabbit-derived antibodies

• Clonality: Both monoclonal (e.g., F-7, E-2) and polyclonal antibodies

• Isotypes: Mouse IgG1 κ is common for monoclonal antibodies

• Applications: Antibodies validated for Western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), immunohistochemistry (IHC), and ELISA

• Species reactivity: Antibodies targeting human, mouse, and rat KLHL36 protein

What validation methods should I look for when selecting a KLHL36 antibody?

When selecting a KLHL36 antibody, look for products validated through:

• Standard validation: Confirming concordance with experimental gene/protein characterization data in databases like UniProtKB/Swiss-Prot.

• Enhanced validation methods:

  • siRNA knockdown validation: Demonstrating decreased antibody staining upon target protein downregulation

  • GFP-tagged cell line validation: Confirming signal overlap between antibody staining and GFP-tagged protein

  • Independent antibodies validation: Comparing staining patterns of multiple antibodies directed toward different epitopes

• Western blot validation: Confirming specific band detection at approximately 70 kDa in relevant lysates

• Tissue screening: Validation across multiple tissues helps ensure specificity in various biological contexts

How can I verify the specificity of my KLHL36 antibody?

To verify KLHL36 antibody specificity:

• Perform knockdown experiments: Use KLHL36 siRNA (e.g., sc-141541 for mouse or sc-93153 for human) to reduce expression and confirm reduced signal intensity in immunoblotting or immunostaining.

• Use positive control lysates: HepG2 and HeLa lysates have been documented as reliable positive controls for KLHL36 detection.

• Epitope mapping: Consider using antibodies targeting different epitopes of KLHL36 to confirm detection of the same protein.

• Test with recombinant protein: Consider using purified recombinant KLHL36 protein as a positive control.

• Cross-reactivity assessment: Test the antibody against related Kelch-like proteins to ensure specificity .

What are the optimal dilutions and conditions for using KLHL36 antibodies in different applications?

Recommended dilutions and conditions vary by application and specific antibody:

• Western Blotting:

  • Typical starting dilutions range from 1:100 to 1:1000

  • For HPA023423: 0.04-0.4 μg/mL

  • Look for a band at approximately 70 kDa

• Immunofluorescence:

  • Starting dilution of 1:50 to 1:200

  • For HPA023423: 0.25-2 μg/mL

• Immunohistochemistry:

  • For HPA023423: 1:200-1:500 dilution

  • Positive controls include HepG2 and HeLa cell lysates

• Immunoprecipitation:

  • Typically 1-2 μg per 100-500 μg of total protein (1 ml of cell lysate)

• ELISA:

  • Starting dilution of 1:30, with a dilution range of 1:30-1:3000

How can I design effective KLHL36 knockdown experiments?

For effective KLHL36 knockdown experiments:

• Selection of knockdown tools:

  • Use validated siRNA such as KLHL36 siRNA (h): sc-93153 or KLHL36 siRNA (m): sc-141541

  • Alternative options include shRNA plasmids (sc-93153-SH for human, sc-141541-SH for mouse)

  • For stable knockdown, consider lentiviral particles (sc-93153-V for human, sc-141541-V for mouse)

• Experimental design:

  • Include appropriate controls (non-targeting siRNA)

  • Verify knockdown efficiency using Western blotting with validated KLHL36 antibodies

  • Use F-7 or E-2 monoclonal antibodies as control antibodies for monitoring KLHL36 expression knockdown

• Phenotypic assessment:

  • Design assays to measure impacts on ubiquitination pathways

  • Assess effects on CUL-3 interaction partners

  • Evaluate changes in protein degradation pathways

How does KLHL36 functionally interact with the CUL-3 ubiquitination pathway?

KLHL36 functions within the CUL-3 ubiquitination pathway through several key mechanisms:

• Complex formation: KLHL36 forms a complex with CUL-3 through its BTB/POZ domain, which facilitates protein binding and dimerization.

• Substrate recognition: The Kelch repeats in KLHL36 likely function as substrate recognition domains, identifying specific proteins for ubiquitination.

• E3 ligase complex: Within this complex, KLHL36 serves as a substrate-specific adapter that helps position target proteins for ubiquitin transfer from E2 ubiquitin-conjugating enzymes.

• Cellular regulation: This interaction impacts cellular processes including cell cycle regulation and signal transduction pathways.

To investigate these interactions experimentally:

• Use co-immunoprecipitation with KLHL36 antibodies to pull down CUL-3 and associated proteins

• Consider proximity ligation assays to visualize KLHL36-CUL-3 interactions in situ

• Employ CRISPR-based approaches to modify interaction domains and assess functional consequences

What are the differences in function between the two isoforms of KLHL36, and how can I distinguish them experimentally?

The two KLHL36 isoforms result from alternative splicing, but their functional differences are not well characterized in the literature. To distinguish and study these isoforms:

• Molecular distinction:

  • Design isoform-specific primers for RT-PCR to quantify expression of each isoform

  • Use antibodies targeting unique regions of each isoform, if available

  • Consider overexpression studies with tagged constructs of each isoform

• Functional analysis:

  • Compare subcellular localization patterns through immunofluorescence

  • Assess differential binding partners through co-immunoprecipitation followed by mass spectrometry

  • Evaluate differential effects on ubiquitination targets

  • Employ isoform-specific siRNAs to determine unique functions

• Experimental approach:

  • Use CRISPR-based genome editing to selectively modify one isoform

  • Consider proteomic approaches to identify isoform-specific post-translational modifications

  • Investigate tissue-specific expression patterns of each isoform

What are common issues with KLHL36 detection in Western blotting, and how can they be resolved?

Common issues in KLHL36 Western blotting include:

• Weak or no signal:

  • Increase antibody concentration (try 0.4 μg/mL for HPA023423)

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

  • Ensure sample contains adequate KLHL36 (use HepG2 or HeLa lysates as positive controls)

  • Check protein transfer efficiency with reversible staining

• Multiple bands or high background:

  • Increase blocking time or concentration

  • Use more stringent washing conditions

  • Try a different antibody (consider F-7 or E-2 monoclonal antibodies)

  • Increase dilution of secondary antibody

• Incorrect molecular weight detection:

  • KLHL36 should appear at approximately 70 kDa

  • Verify sample preparation conditions (reducing vs. non-reducing)

  • Check for post-translational modifications that may alter migration

  • Confirm specificity with KLHL36 knockdown controls

How can I optimize immunofluorescence staining with KLHL36 antibodies?

For optimal immunofluorescence staining with KLHL36 antibodies:

• Fixation optimization:

  • Test different fixation methods (4% paraformaldehyde vs. methanol)

  • Consider antigen retrieval methods if signal is weak

  • Optimize fixation time to preserve epitope accessibility

• Antibody considerations:

  • Use recommended dilutions (0.25-2 μg/mL for HPA023423)

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

  • Use monoclonal antibodies (F-7 or E-2) for potentially cleaner signal

• Signal enhancement strategies:

  • Implement signal amplification systems if needed

  • Optimize permeabilization conditions to improve antibody access

  • Consider co-staining with CUL-3 or other known interacting partners

• Controls:

  • Include KLHL36 knockdown cells as negative controls

  • Use cells with known KLHL36 expression (e.g., HeLa) as positive controls

  • Consider GFP-tagged KLHL36 expression as a validation approach

Beyond antibodies, what tools are available for manipulating KLHL36 expression in experimental systems?

Several molecular tools are available for KLHL36 research:

• Gene silencing tools:

  • siRNA for transient knockdown (sc-93153 for human, sc-141541 for mouse)

  • shRNA plasmids for longer-term knockdown (sc-93153-SH for human, sc-141541-SH for mouse)

  • Lentiviral particles for stable integration (sc-93153-V for human, sc-141541-V for mouse)

• CRISPR-based tools:

  • CRISPR/Cas9 KO plasmids for gene knockout (sc-413245 for human, sc-433396 for mouse)

  • HDR plasmids for homology-directed repair (sc-413245-HDR for human, sc-433396-HDR for mouse)

  • Double Nickase plasmids for reduced off-target effects (sc-413245-NIC for human, sc-433396-NIC for mouse)

• Gene activation tools:

  • CRISPR Activation Plasmids for gene upregulation (sc-413245-ACT for human, sc-433396-ACT for mouse)

  • Lentiviral Activation Particles (sc-413245-LAC for human, sc-433396-LAC for mouse)

• Recombinant proteins:

  • Full-length recombinant KLHL36 with various tags for in vitro studies

  • Specific domains for structure-function relationship studies

How can I design experiments to identify novel substrates of KLHL36 in the ubiquitination pathway?

To identify novel KLHL36 substrates:

• Protein interaction screening:

  • Perform immunoprecipitation with KLHL36 antibodies followed by mass spectrometry

  • Use yeast two-hybrid screening with Kelch domains as bait

  • Consider BioID or proximity labeling approaches to identify proximal proteins

• Ubiquitination assays:

  • Conduct in vitro ubiquitination assays with recombinant KLHL36-CUL3 complexes

  • Perform global ubiquitinome analysis comparing wild-type and KLHL36 knockout cells

  • Use ubiquitin remnant profiling to identify differentially ubiquitinated proteins

• Functional validation:

  • Confirm direct interaction between KLHL36 and putative substrates

  • Demonstrate ubiquitin-dependent degradation of candidate substrates

  • Perform domain mapping to identify specific interaction regions

• Systems biology approaches:

  • Integrate transcriptomic and proteomic data from KLHL36 perturbation experiments

  • Consider computational prediction of potential substrates based on structural features

  • Examine protein stability changes upon KLHL36 manipulation