KIAA1468 Antibody

What is KIAA1468 protein and what are its cellular functions?

KIAA1468, also known as RELCH (RAB11-binding protein RELCH), is a newly identified Rab11 effector protein with significant roles in cholesterol transport. The protein:

• Functions as a membrane tether between recycling endosomes (REs) and the trans-Golgi network (TGN)

• Binds to TGN-localized oxysterol-binding protein (OSBP)

• Promotes OSBP-dependent nonvesicular cholesterol transport from REs to the TGN

• Contains structural elements including 3 HEAT repeats and 1 LisH domain

The protein has a calculated molecular weight of 139 kDa, though Western blot typically shows observed molecular weight between 100-135 kDa . This discrepancy may reflect post-translational modifications or protein processing events.

What experimental applications are KIAA1468 antibodies validated for?

KIAA1468 antibodies have been validated for multiple experimental applications with specific recommended protocols for each:

For optimal results, each application requires specific sample preparation methods and antibody concentration optimization based on the experimental system .

What are the best tissue and cell types for studying KIAA1468 expression?

Based on antibody validation data, the following tissue and cell types show reliable KIAA1468 detection:

• Tissues with strong expression: Mouse brain and kidney tissues consistently show positive staining across multiple antibody products

• Cell lines: Human U-2 OS cells show cytoplasmic localization of KIAA1468

• Additional validated human samples: Pancreatic tissue, RT-4 and U-251 MG cell lysates have been validated as positive controls

When selecting experimental models, researchers should consider both the species compatibility of their selected antibody and the relative expression levels in different tissues. For human samples, kidney tissue sections have demonstrated consistent staining patterns suitable for IHC applications .

What controls should be included when using KIAA1468 antibodies?

Proper experimental controls are essential for reliable KIAA1468 antibody applications:

Positive controls:

• Mouse brain tissue lysate for WB and IP applications

• Mouse kidney tissue for IHC applications

• Human U-2 OS cells for ICC/IF applications

Negative controls:

• Primary antibody omission control

• Isotype control (Rabbit IgG at equivalent concentration)

• Peptide competition assay using the immunizing peptide

Validation controls:

• siRNA knockdown or CRISPR knockout of KIAA1468 (genetic validation)

• Comparison with independent antibodies targeting different epitopes of KIAA1468

• Orthogonal validation comparing protein expression with transcript levels

The Human Protein Atlas project has established validation standards for antibodies against KIAA1468, including standard and enhanced validation methods that researchers should reference when selecting controls .

How should KIAA1468 antibody dilutions be optimized for specific experimental applications?

Optimizing antibody dilutions requires systematic titration based on signal-to-noise ratio evaluation:

Western Blot optimization:

• Begin with the manufacturer's recommended range (1:500-1:1000)

• Perform a dilution series (e.g., 1:250, 1:500, 1:1000, 1:2000)

• Assess specific band intensity at 100-135 kDa relative to background

• Validate specificity through lysates with known expression levels (e.g., mouse brain tissue)

• Consider secondary antibody optimization in parallel

IHC optimization:

• Start within recommended dilution range (1:50-1:500)

• Test multiple antigen retrieval conditions:

  • TE buffer pH 9.0 (primary recommendation)

  • Citrate buffer pH 6.0 (alternative method)

• Perform serial dilutions on positive control tissue (mouse kidney)

• Score staining intensity, specificity, and background

• Incorporate tissue-specific negative controls to establish background threshold

IP optimization:

• Begin with minimum recommended antibody amount (0.5 μg per mg of lysate)

• Increase antibody amount incrementally if initial pull-down is insufficient

• Optimize binding conditions (time, temperature, buffer composition)

• Validate specificity through Western blot of immunoprecipitated material

For all applications, the principle "use the highest dilution that maintains specific signal" applies to minimize background and cross-reactivity issues.

What are the best validation strategies to confirm KIAA1468 antibody specificity?

Comprehensive validation of KIAA1468 antibodies should employ multiple complementary approaches:

Enhanced validation methods:

• Genetic validation: siRNA knockdown or CRISPR/Cas9 knockout of KIAA1468, demonstrating decreased signal intensity

• Independent antibody validation: Compare staining patterns using antibodies targeting different epitopes of KIAA1468

  • Proteintech 18993-1-AP (peptide-derived immunogen)

  • Abcam ab122612 (amino acids 1115-1194)

  • Abcam ab122576 (amino acids 500-650)

• Orthogonal validation: Correlation of protein expression with transcript analysis data

• Recombinant expression validation: Overexpression of tagged KIAA1468 to confirm antibody detection

Technical validation:

• Peptide competition assay: Pre-incubation with immunizing peptide should abolish specific signal

• Western blot analysis: Confirmation of expected molecular weight (100-135 kDa)

• Cross-species reactivity: Consistent detection in homologous proteins across validated species (human, mouse, rat)

These validation approaches align with Human Protein Atlas standards, which recommend combining standard validation (based on UniProtKB/Swiss-Prot data) with enhanced validation methods for comprehensive antibody qualification .

How can subcellular localization of KIAA1468 be accurately determined using immunofluorescence techniques?

Accurate subcellular localization requires careful optimization of immunofluorescence protocols:

Sample preparation optimization:

• Test multiple fixation methods:

  • 4% paraformaldehyde (10-15 minutes)

  • Methanol fixation (-20°C, 10 minutes)

  • Combined PFA/Triton X-100 treatment

• Optimize permeabilization conditions:

  • 0.1-0.5% Triton X-100

  • 0.1-0.5% Saponin for membrane preservation

• Employ antigen retrieval if necessary

Co-localization analysis:

• Select markers for relevant subcellular compartments:

  • Rab11 (recycling endosomes)

  • TGN46 or Golgin-97 (trans-Golgi network)

  • OSBP (oxysterol-binding protein)

• Use fluorophore combinations with minimal spectral overlap

• Acquire images using confocal microscopy with appropriate controls

• Quantify co-localization using Pearson's or Mander's coefficient

Methodological considerations:

• Use validated cell lines (e.g., U-2 OS) showing cytoplasmic staining pattern

• Implement Z-stack acquisition to capture complete subcellular distribution

• Include positive controls for known KIAA1468 cellular functions (e.g., cholesterol transport assays)

• Confirm specificity through genetic manipulation experiments

These approaches will help establish the functional localization of KIAA1468 at the interface between recycling endosomes and trans-Golgi network, consistent with its role in membrane tethering and cholesterol transport .

How do different tissue fixation and preparation methods affect KIAA1468 antibody performance in IHC?

Tissue fixation and processing significantly impact KIAA1468 antibody performance in immunohistochemistry:

Fixation method comparison:

Antigen retrieval optimization:

• Heat-induced epitope retrieval (HIER):

  • TE buffer pH 9.0 (primary recommendation)

  • Citrate buffer pH 6.0 (alternative method)

  • Optimization of heating conditions (95-100°C for 10-30 minutes)

• Enzymatic retrieval methods:

  • Proteinase K digestion (alternative for resistant tissues)

  • Trypsin digestion (limited data for KIAA1468)

Tissue-specific considerations:

• Highly vascularized tissues may require additional blocking steps

• Mouse kidney tissue serves as reliable positive control across fixation methods

• Tissue thickness (4-5 μm optimal) affects antibody penetration and signal intensity

• Post-fixation storage conditions impact epitope preservation (store at 4°C and use within 1 week)

Researchers should validate their specific tissue preparation methodology through side-by-side comparisons using positive control tissues before proceeding with experimental samples.

What computational approaches are being developed for designing antibodies against challenging epitopes like KIAA1468?

Recent advances in computational antibody design offer promising approaches for generating antibodies against challenging targets:

Deep learning model applications:

• Generative models for antibody sequences:

  • Training on datasets of human antibodies with computational developability criteria

  • Generation of variable region sequences with desired physicochemical properties

  • Production of antibody libraries with "medicine-likeness" and high humanness

• Binding specificity engineering:

  • Identification of distinct binding modes associated with specific ligands

  • Computational design of antibodies with customized specificity profiles

  • Generation of antibodies with either high specificity for particular targets or cross-specificity for multiple targets

Implementation methodology:

• Begin with high-throughput sequencing of antibody repertoires

• Apply biophysics-informed modeling to identify potential binding modes

• Optimize energy functions associated with desired binding profiles

• Generate novel sequences through computational methods

• Validate experimentally through phage display or direct protein production

These computational approaches offer several advantages for KIAA1468 antibody development:

• Ability to design antibodies beyond those identified through experimental selection

• Creation of antibodies with precisely defined specificity profiles

• Overcoming limitations of traditional library size and selection biases

• Potential for designing antibodies against epitopes that are difficult to access experimentally

Recent studies have demonstrated successful experimental validation of computationally designed antibodies, showing high expression, monomer content, thermal stability, and reduced non-specific binding .

How can researchers interpret conflicting KIAA1468 expression data between antibody-based methods and transcript analysis?

Resolving discrepancies between protein and transcript data requires systematic investigation:

Common sources of discrepancy:

• Post-transcriptional regulation:

  • miRNA-mediated suppression of protein synthesis

  • Variations in translational efficiency

  • Protein stability and turnover differences

• Technical considerations:

  • Antibody specificity issues or cross-reactivity

  • Epitope masking in certain tissue/fixation conditions

  • Transcript variant detection differences in RNA analyses

Methodological approach to resolution:

• Multi-antibody validation:

  • Test independent antibodies targeting different KIAA1468 epitopes

  • Compare staining patterns between antibodies (18993-1-AP, ab122612, ab122576, HPA039708)

  • Identify consistent expression patterns across multiple antibodies

• Orthogonal validation:

  • Correlate protein expression with transcript data from multiple sources

  • Employ protein mass spectrometry as independent validation

  • Utilize genetic manipulation (knockdown/overexpression) with monitoring of both protein and transcript levels

• Context-specific evaluation:

  • Consider tissue-specific post-transcriptional regulation

  • Evaluate subcellular localization patterns

  • Assess potential protein modification states affecting detection

Quantitative analysis framework:

• Establish standardized scoring systems for both protein and transcript expression

• Apply statistical methods to identify significant discrepancies

• Develop tissue-specific correction factors if systematic differences are observed

• Consider biological context (e.g., KIAA1468's role in cholesterol transport may affect expression in lipid-rich tissues)

The Human Protein Atlas project employs standardized orthogonal validation approaches that can serve as a methodological template for resolving such discrepancies .

What are the best strategies for studying KIAA1468 protein interactions with partners like Rab11 and OSBP?

Investigating KIAA1468 protein interactions requires specialized methodological approaches:

Immunoprecipitation-based methods:

• Co-immunoprecipitation (Co-IP):

  • Use validated KIAA1468 antibodies (18993-1-AP, 0.5-4.0 μg for 1.0-3.0 mg lysate)

  • Verify antibody suitability for IP in mouse brain tissue

  • Include appropriate controls (IgG control, lysate input)

  • Detect interacting partners (Rab11, OSBP) by Western blot

• Proximity-dependent labeling:

  • BioID or TurboID fusion with KIAA1468 to identify proximal proteins

  • APEX2 proximity labeling for temporal interaction dynamics

  • Mass spectrometry analysis of labeled proteins

Fluorescence microscopy approaches:

• Fluorescence resonance energy transfer (FRET):

  • Create fluorophore-tagged constructs of KIAA1468 and interaction partners

  • Measure energy transfer indicating proximity (<10 nm)

  • Employ acceptor photobleaching or fluorescence lifetime imaging

• Co-localization analysis:

  • Dual immunofluorescence using validated antibodies

  • Super-resolution microscopy for detailed interaction sites

  • Live-cell imaging to track dynamic interactions

Functional interaction assays:

• Cholesterol transport assays:

  • Measure KIAA1468-dependent cholesterol transfer between REs and TGN

  • Assess effects of Rab11 or OSBP manipulation on transport efficiency

  • Utilize fluorescent cholesterol analogs for real-time tracking

• Membrane tethering assays:

  • In vitro reconstitution of membrane tethering

  • Liposome-based assays measuring KIAA1468-dependent membrane association

  • Effects of mutations in key domains (HEAT repeats, LisH domain) on tethering activity

These methodological approaches can be combined to build a comprehensive understanding of how KIAA1468 functions at the molecular level in membrane tethering and cholesterol transport processes in collaboration with its binding partners Rab11 and OSBP.

What emerging technologies are improving antibody reliability for complex targets like KIAA1468?

Recent technological advances are addressing longstanding challenges in antibody reliability:

Validation technologies:

• Enhanced genetic validation:

  • CRISPR knockout cell lines as definitive negative controls

  • Inducible expression systems for titratable positive controls

  • Genome-edited cell lines expressing tagged endogenous proteins

• Advanced imaging validation:

  • Super-resolution microscopy for precise co-localization analysis

  • Expansion microscopy for improved spatial resolution of interactions

  • Single-molecule tracking for dynamic protein behavior

Production improvements:

• Computational antibody design:

  • Deep learning approaches generating antibodies with improved specificity

  • Biophysics-informed modeling to predict binding modes

  • In silico optimization of developability attributes

• Recombinant antibody technologies:

  • Single B-cell sequencing for monoclonal antibody discovery

  • Phage display with high-throughput sequencing for specificity analysis

  • Site-specific conjugation for consistent antibody performance

These technological developments promise to improve the reliability, reproducibility, and specificity of antibodies for complex targets like KIAA1468, addressing the ongoing reproducibility challenges in life science research .

How can researchers best integrate KIAA1468 antibody data with broader proteomic and genomic datasets?

Integrative approaches to data analysis enhance the value of antibody-based research:

Data integration strategies:

• Multi-omics correlation analysis:

  • Compare antibody-based protein detection with transcriptomic data

  • Correlate KIAA1468 expression with interacting partners (Rab11, OSBP)

  • Integrate with lipid profiling data relevant to cholesterol transport function

• Pathway and network analysis:

  • Position KIAA1468 within membrane trafficking networks

  • Identify co-regulated genes in cholesterol homeostasis pathways

  • Map protein-protein interactions through integrative database analysis

Methodological frameworks:

• Standardized data reporting:

  • Document complete antibody metadata (catalog number, lot, dilution, conditions)

  • Report validation methods employed

  • Share raw imaging and quantification data

• Computational analysis pipelines:

  • Employ machine learning for pattern recognition across datasets

  • Develop tissue-specific expression models

  • Create predictive models for KIAA1468 function in different cellular contexts