LRP2 Antibody

What is LRP2 and why is it important in research?

LRP2, also known as megalin or gp330, is a large transmembrane protein (522 kDa) belonging to the low-density lipoprotein receptor family. It functions as a multi-ligand endocytic receptor critical for the reabsorption of numerous molecules in the proximal tubule of the kidney and other tissues. Research significance stems from its involvement in kidney disease, Donnai-Barrow syndrome (DBS), and potential roles in Alzheimer's disease pathogenesis. The protein has a large amino-terminal extracellular domain, a single transmembrane domain, and a short carboxy-terminal cytoplasmic tail. Its extracellular domain binds diverse macromolecules including albumin, apolipoproteins B and E, and lipoprotein lipase .

What are the common applications for LRP2 antibodies in research?

LRP2 antibodies are employed in multiple research applications, with different optimization requirements for each:

These applications have been validated across human, mouse, and rat samples, with cross-reactivity varying by specific antibody clone .

How should LRP2 antibodies be stored to maintain optimal activity?

Most LRP2 antibodies require storage at -20°C for long-term stability. The typical storage buffer consists of PBS (pH 7.2-7.4) with 0.02% sodium azide and 50% glycerol. These conditions prevent freeze-thaw damage while maintaining antibody functionality. Antibodies are generally stable for one year after shipment when stored properly. For smaller volume antibodies (typically 20μl sizes), manufacturers often include 0.1% BSA in the formulation to prevent protein loss through adsorption to container walls. Aliquoting is not typically necessary for -20°C storage when glycerol is present, but is recommended when antibodies are stored for extended periods .

What control samples are recommended when using LRP2 antibodies?

For rigorous experimental design, the following controls are recommended:

• Positive tissue controls: Kidney tissue (human, mouse, rat) shows strong expression, particularly in proximal tubules

• Negative controls: Tissue known to lack LRP2 expression, or use of isotype-matched irrelevant antibodies

• Knockout/knockdown validation: Several LRP2 antibodies have been validated in KO/KD systems as indicated in publications

• Peptide competition assay: Pre-incubation with the immunogenic peptide should abolish specific staining

• Multiple antibody verification: Using antibodies recognizing different epitopes provides stronger evidence of specificity

How can epitope-specific LRP2 antibodies be leveraged to study different disease mechanisms?

The selection of epitope-specific antibodies is crucial for studying particular disease mechanisms, as different epitopes on LRP2 are implicated in various pathologies:

• N-terminal domain antibodies: Nine of ten anti-LRP2 nephropathy patient sera specifically recognized the N-terminal set of seven LA repeats from LRP2, making antibodies to this region valuable for studying autoimmune mechanisms .

• C-terminal antibodies: Useful for studying endocytosis mechanisms as the C-terminal cytoplasmic region interacts with endocytic machinery.

• Domain-specific targeting:

  • LA1-7 (N-terminal): Implicated in autoimmune disease

  • LA8-15: Associated with ligand binding

  • LA16-25: Additional autoepitopes in rheumatoid arthritis

  • LA26-32: Contains autoepitopes in multiple conditions

Researchers should select antibodies targeting specific domains based on the disease mechanism being studied. For example, in anti-LRP2 nephropathy research, antibodies to F3, F4, F5, and F6 fragments are particularly relevant as the presence of autoantibodies to these regions correlates with proteinuria in rheumatoid arthritis patients .

What methodological approaches can resolve the discrepancy between predicted and observed molecular weights of LRP2 in Western blots?

LRP2 presents a common challenge in Western blotting where the observed molecular weight (~280-330 kDa) often differs from the predicted weight (522 kDa). This discrepancy can be addressed through several methodological approaches:

• Protein extraction optimization:

  • Use of specialized lysis buffers containing protease inhibitor cocktails to prevent fragmentation

  • Gentle extraction methods at 4°C to minimize proteolysis

  • Inclusion of N-ethylmaleimide to prevent artifactual disulfide bond formation

• Electrophoresis conditions:

  • Low percentage (3-5%) polyacrylamide gels for better resolution of high molecular weight proteins

  • Gradient gels (3-8%) to improve separation

  • Extended running times at lower voltages to allow complete migration of large proteins

• Alternative analytical approaches:

  • Mass spectrometry analysis to confirm protein identity and determine fragmentation patterns

  • Native gel electrophoresis to preserve protein integrity

  • Immunoprecipitation followed by Western blotting to enrich target protein

The 280-330 kDa bands frequently observed represent proteolytic fragments of the large glycoprotein, a phenomenon previously documented in the literature where LRP2 was initially known as gp330 due to its migration at approximately 330 kDa position .

How do pH-dependent conformational changes in LRP2 affect antibody binding and experimental design?

Recent cryo-electron microscopy studies have revealed that LRP2 undergoes significant pH-dependent conformational changes that are crucial for its function in ligand binding at the cell surface (neutral pH) versus ligand shedding in the endosome (acidic pH) . These conformational changes have important implications for antibody-based experiments:

• Buffer considerations for immunoprecipitation:

  • At pH 7.4: LRP2 adopts an "open" conformation favorable for ligand binding

  • At pH 5.5-6.0: LRP2 adopts a "closed" conformation associated with ligand release

• Epitope accessibility variations:

  • Certain epitopes may be masked or exposed depending on pH conditions

  • Conformational antibodies may show pH-dependent binding patterns

• Live cell imaging considerations:

  • Antibodies recognizing pH-sensitive epitopes may show differential binding during endocytosis

  • Dual labeling with pH-insensitive antibodies recommended for tracking studies

• Experimental design recommendations:

  • Include pH controls in binding experiments

  • Consider pH effects when interpreting negative results

  • Test antibody binding efficiency at both neutral and acidic pH conditions

  • For endocytosis studies, select antibodies recognizing pH-stable epitopes

These pH-dependent conformational changes are governed by pH-sensitive sites at both homodimer and intra-protomer interfaces, which should be considered when analyzing antibody binding data in different subcellular compartments .

What approaches can distinguish between LRP2 autoantibodies and exogenous anti-LRP2 antibodies in biological samples?

In clinical research involving anti-LRP2 nephropathy or autoimmune conditions, distinguishing between endogenous autoantibodies and research antibodies is methodologically challenging but crucial:

• Isotype-specific secondary antibodies:

  • Human autoantibodies are often IgG4 subclass in anti-LRP2 nephropathy

  • Most research antibodies are rabbit or mouse-derived

  • Use species-specific secondary antibodies for differential detection

• Epitope mapping strategies:

  • Patient autoantibodies typically recognize the N-terminal domain (LA1-7)

  • Map the specific binding regions using recombinant LRP2 fragments

  • Compare reactivity patterns between patient samples and research antibodies

• Validated detection protocols:

  • For clinical samples: Pre-absorption against irrelevant antigens to reduce background

  • Western blotting using recombinant LRP2 fragments (LA1-7, LA8-15, LA16-25, and LA26-32)

  • Competitive binding assays to assess epitope overlap

• Differentiation table for result interpretation:

This approach is particularly important when studying anti-LRP2 nephropathy, which affects approximately 1.3% of elderly patients with kidney disease and may be associated with B-cell lymphoproliferative disorders .

How can researchers optimize immunohistochemical detection of LRP2 in tissues with both tubular and glomerular expression?

Detecting LRP2 across different kidney structures presents technical challenges due to varying expression levels (high in proximal tubules, lower in podocytes). A comprehensive optimization protocol includes:

• Antigen retrieval optimization:

  • Test both heat-induced epitope retrieval methods:

    • TE buffer (pH 9.0) - often superior for LRP2

    • Citrate buffer (pH 6.0) - alternative option

  • Optimize retrieval duration (15-30 minutes)

• Primary antibody strategy:

  • For comprehensive detection: Use antibodies targeting conserved epitopes

  • For structure-specific analysis: Select domain-specific antibodies

  • Consider antibody cocktails for maximum sensitivity

• Signal amplification methods:

  • Polymer-based detection systems for routine IHC

  • Tyramide signal amplification for low-expression regions (glomeruli)

  • Quantum dot labeling for multi-epitope visualization

• Tissue-specific considerations:

  • Use sequential sections to compare staining patterns

  • Include both normal and pathological samples

  • Implement proper positive controls (kidney tissue) and negative controls

• Visualization optimization:

  • Confocal microscopy for co-localization studies

  • Digital quantification using normalized reference standards

  • Counter-staining with structure-specific markers (WT-1 for podocytes)

This approach acknowledges the controversial nature of podocyte LRP2 expression while providing reliable detection methods. Recent evidence suggests human podocytes do express LRP2, though at lower levels than proximal tubular cells, which explains the segmental pattern of deposits seen in anti-LRP2 nephropathy .

What protein extraction methods are optimal for preserving LRP2 integrity in Western blot applications?

LRP2's large size (522 kDa) and susceptibility to proteolysis require specialized extraction methods:

• Recommended extraction buffer composition:

  • Base buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl

  • Detergents: 1% NP-40 or 0.5% Triton X-100

  • Protease inhibitors: Complete cocktail plus 5 mM EDTA, 5 mM EGTA

  • Phosphatase inhibitors: 10 mM NaF, 1 mM Na3VO4

  • Additional protectants: 1 mM PMSF, 10 mM N-ethylmaleimide

• Extraction procedure optimization:

  • Maintain samples at 4°C throughout processing

  • Use gentle mechanical disruption (Dounce homogenizer)

  • Limit sonication to brief pulses to avoid protein degradation

  • Centrifuge at 14,000g for 15 minutes to remove debris

• Sample preparation for electrophoresis:

  • Avoid boiling samples (incubate at 37°C for 30 minutes instead)

  • Use reducing conditions (5% β-mercaptoethanol)

  • Load adequate protein (50-100 μg total protein per lane)

  • Use low percentage (3-5%) or gradient gels for separation

These methods significantly improve detection of full-length LRP2 while minimizing proteolytic fragments that complicate result interpretation .

How can researchers differentiate between specific and non-specific binding in LRP2 immunostaining protocols?

Establishing specificity in LRP2 immunostaining is essential given the protein's wide distribution and molecular complexity:

• Essential controls for specificity validation:

  • Peptide competition: Pre-incubation with immunizing peptide should abolish staining

  • Knockout/knockdown tissues: Complete absence or reduction of signal

  • Multiple antibody approach: Concordant results with antibodies to different epitopes

  • Isotype controls: Matched irrelevant antibodies to assess non-specific binding

• Technical measures to reduce background:

  • Optimal blocking (5% BSA or 10% normal serum from secondary antibody species)

  • Inclusion of 0.1-0.3% Triton X-100 for membrane permeabilization

  • Endogenous peroxidase blocking (3% H₂O₂ for 10 minutes)

  • Endogenous biotin blocking if avidin-biotin detection systems are used

• Pattern recognition for result interpretation:

  • Specific LRP2 staining: Brush border of proximal tubules (strong), podocytes (weaker, segmental)

  • Non-specific patterns: Diffuse cytoplasmic staining, nuclear staining, or staining in tissues known to lack LRP2

• Quantitative assessment of specificity:

  • Signal-to-noise ratio measurements

  • Comparison with established reference standards

  • Correlation with mRNA expression data

  • Western blot validation of antibody specificity

The most convincing evidence comes from showing that the antibody recognizes the same pattern as seen in human anti-LRP2 nephropathy, where LRP2 colocalizes with IgG in tubular immune deposits but not in control specimens .

What methods can be used to validate novel LRP2 antibodies for research applications?

Comprehensive validation of new LRP2 antibodies should follow a multi-step approach:

• Epitope characterization:

  • Epitope mapping using overlapping peptide arrays

  • Competition assays with established antibodies

  • Structural prediction of epitope accessibility

  • Cross-reactivity assessment across species (human, mouse, rat)

• Biochemical validation:

  • Western blot against recombinant LRP2 fragments

  • Immunoprecipitation followed by mass spectrometry

  • ELISA using purified LRP2 protein

  • Surface plasmon resonance for binding kinetics

• Cellular validation:

  • Immunocytochemistry on cells with known LRP2 expression

  • Flow cytometry on kidney-derived cell lines

  • siRNA/CRISPR knockdown to confirm specificity

  • Comparison with mRNA expression (qPCR, in situ hybridization)

• Tissue validation matrix:

• Functional validation:

  • Antibody effect on LRP2-dependent endocytosis

  • Ability to recognize native vs. denatured protein

  • pH-dependent binding characteristics

  • Cross-reactivity with related LDL receptor family members

This systematic approach ensures antibodies are suitable for their intended applications and minimizes potential false-positive or false-negative results in research settings .

How should researchers design experiments to study the role of LRP2 in autoimmune kidney diseases?

Investigating LRP2's role in autoimmune kidney diseases requires a multifaceted experimental approach:

• Patient sample analysis:

  • Serum screening for anti-LRP2 autoantibodies using recombinant fragments

  • Epitope mapping to identify pathogenic autoantibody targets

  • Isotype and subclass determination (IgG4 predominance in anti-LRP2 nephropathy)

  • Correlation of autoantibody titers with clinical parameters (proteinuria, renal function)

• Tissue examination protocols:

  • Immunofluorescence co-localization of LRP2 with immune deposits

  • Dual staining for LRP2 and IgG in kidney biopsies

  • Electron microscopy to characterize deposit ultrastructure

  • Laser capture microdissection of affected areas for molecular analysis

• Functional studies:

  • In vitro assessment of autoantibody effects on LRP2-mediated endocytosis

  • Cell culture models using patient-derived autoantibodies

  • Complement activation assays to evaluate immune complex pathogenicity

  • Protein reabsorption assays to measure functional impairment

• Animal models:

  • Passive transfer of patient-derived antibodies

  • Active immunization with LRP2 fragments to induce autoimmunity

  • Transgenic models expressing human LRP2

  • Therapeutic intervention studies

• Relationship to other conditions:

  • Screening for LRP2 autoantibodies in rheumatoid arthritis (87% prevalence)

  • Assessment in B-cell lymphoproliferative disorders

  • Evaluation in elderly patients with unexplained proteinuria

This comprehensive approach has revealed that anti-LRP2 nephropathy may be underdiagnosed, affecting approximately 1.3% of elderly patients with kidney disease and potentially associated with B-cell lymphoproliferative disorders .

What considerations are important when using LRP2 antibodies for studying protein trafficking and endocytosis?

LRP2's function in endocytosis requires specialized experimental approaches:

• Antibody selection for trafficking studies:

  • Extracellular domain antibodies: For surface binding and internalization tracking

  • Intracellular domain antibodies: For cytoplasmic tail interactions

  • Non-function-blocking antibodies: For passive tracking without interfering with endocytosis

  • pH-insensitive epitope antibodies: For tracking through acidifying compartments

• Live cell imaging considerations:

  • Direct fluorophore conjugation to minimize size effects

  • Fab fragments to reduce crosslinking

  • Pulse-chase protocols for tracking internalization kinetics

  • Photoactivatable or pH-sensitive fluorophores for compartment-specific visualization

• Co-localization studies design:

  • Markers for different endocytic compartments:

    • Clathrin: Initial endocytosis

    • EEA1: Early endosomes

    • Rab7: Late endosomes

    • LAMP1: Lysosomes

    • Rab11: Recycling endosomes

  • Fixed time-point series to capture trafficking dynamics

  • Super-resolution microscopy for precise localization

• Functional endocytosis assays:

  • Fluorescently-labeled LRP2 ligands (albumin, apolipoprotein B, vitamin-binding proteins)

  • Biotinylation-based internalization assays

  • TIRF microscopy for surface dynamics

  • Pulse-chase biochemical fractionation

These approaches should account for LRP2's pH-dependent conformational changes, which are critical for its function in ligand binding at the cell surface (pH 7.4) versus ligand shedding in the endosome (pH 5.5-6.0) .

How can LRP2 antibodies be effectively used to investigate potential roles in neurodegenerative diseases?

LRP2's emerging role in neurodegenerative diseases, particularly Alzheimer's disease, necessitates specialized research approaches:

• CNS-specific expression analysis:

  • Immunohistochemistry of choroid plexus epithelium (primary CNS expression site)

  • Single-cell RNA sequencing correlation with protein expression

  • Comparison between normal and disease-state tissues

  • Age-dependent expression profiling

• Blood-brain barrier studies:

  • Co-localization with blood-brain barrier markers

  • Transcytosis assays for Aβ peptides and apoE

  • In vitro models using brain microvascular endothelial cells

  • Transport studies with labeled peptides ± LRP2 antibodies

• Interaction studies with AD-associated proteins:

  • Co-immunoprecipitation with:

    • Amyloid-β peptides (Aβ40, Aβ42)

    • Apolipoprotein E (particularly apoE4)

    • Clusterin/apoJ

    • Tau protein

  • Proximity ligation assays for in situ interaction detection

  • Surface plasmon resonance for binding kinetics

• Functional analysis protocols:

  • Receptor-mediated clearance assays with/without blocking antibodies

  • Transgenic animal models with conditionally modulated LRP2 expression

  • CSF biomarker correlation with LRP2 function

  • siRNA knockdown effects on amyloid processing

LRP2 polymorphisms have been associated with Alzheimer's disease susceptibility, and studies show its involvement in clearing Aβ40 and Aβ42 peptides across the blood-brain barrier. Similarly, clusterin/apoJ, which associates with AD in genome-wide association studies, is cleared by LRP2 .

What experimental considerations are important for studying LRP2 in homodimeric versus monomeric states?

Recent cryo-electron microscopy studies have revealed that LRP2 functions as a homodimer, with important implications for experimental design:

• Sample preparation for preserving dimeric structures:

  • Mild detergents: digitonin (0.1%) or LMNG (0.01%)

  • Crosslinking approaches: BS3 or DSS crosslinkers at controlled concentrations

  • Native extraction conditions: physiological pH and ionic strength

  • Stabilization with receptor-associated proteins

• Analytical techniques for dimer detection:

  • Blue native PAGE for intact complex separation

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Chemical crosslinking followed by mass spectrometry (XL-MS)

  • Single-molecule imaging techniques

• Functional distinction assays:

  • Ligand binding studies with monomeric vs. dimeric preparations

  • Endocytosis rates comparison between states

  • pH-dependent conformational change assessment

  • Mutagenesis of dimer interface residues

• Pathogenic variant analysis:

  • Investigation of LRP2 variants at dimer interfaces

  • Correlation of dimerization defects with disease phenotypes

  • Structural modeling of variant effects on assembly

  • Rescue experiments to restore dimerization

Studies suggest that a subset of LRP2 deleterious missense variants in humans appear to impair homodimer assembly, potentially explaining their pathogenic mechanisms. The conformational transformation of the LRP2 homodimer is governed by pH-sensitive sites at both homodimer and intra-protomer interfaces, which should be considered in experimental designs .

How should researchers interpret conflicting results regarding LRP2 expression in podocytes?

The expression of LRP2 in human podocytes has been controversial, with conflicting results in the literature. A systematic approach to resolving these contradictions includes:

• Methodological comparison:

  • Detection methods used (IHC, IF, WB, PCR, RNA-seq)

  • Antibody epitopes (N-terminal, C-terminal, internal domains)

  • Sample preparation techniques (fixation methods, antigen retrieval)

  • Detection sensitivity (conventional vs. amplification systems)

• Species-specific considerations:

  • Rat: Well-established expression in podocytes (Heymann nephritis model)

  • Human: Historically reported as negative, but recent evidence supports low-level expression

  • Mouse: Variable reports depending on detection method

• Reconciliation of contradictory data:

  • Expression level differences: Proximal tubules (high) vs. podocytes (low)

  • Developmental regulation: Different expression patterns during development

  • Pathological induction: Upregulation in disease states

  • Technical sensitivity: Newer methods detect previously undetectable expression

• Consensus interpretation:

  • Human podocytes express LRP2 at lower levels than proximal tubular cells

  • This explains the segmental pattern of deposits seen in anti-LRP2 nephropathy

  • Expression may be upregulated in pathological conditions

  • Detection requires high-sensitivity methods with appropriate controls

This interpretation is supported by recent findings showing LRP2 colocalization with podocyte markers such as WT-1, the presence of LRP2 in segmental immune deposits in the subepithelial space, and its demonstrated role in agalsidase uptake by human podocytes in Fabry disease .

How can researchers differentiate between proteolytic fragmentation and alternative isoforms when analyzing LRP2 Western blot data?

The complexity of LRP2 Western blot patterns requires careful analysis to distinguish true biological variations from technical artifacts:

• Methodological approach to differentiation:

  • Size comparison: Full-length LRP2 (522 kDa) vs. common fragments (280-330 kDa)

  • Multiple antibody analysis: N-terminal vs. C-terminal antibodies

  • RNA analysis: RT-PCR with primers spanning potential splice junctions

  • Mass spectrometry: Peptide coverage mapping across the protein sequence

• Proteolytic fragmentation characteristics:

  • Sample-preparation dependent: More fragments with harsh extraction

  • Protease inhibitor-sensitive: Reduced with comprehensive inhibitor cocktails

  • Time and temperature dependent: Increases with storage time and temperature

  • Yields predictable fragments based on known protease cleavage sites

• Alternative isoform indicators:

  • Consistent appearance regardless of extraction method

  • Reproducible across multiple samples and experiments

  • Correlation with mRNA splice variants

  • Tissue-specific or condition-specific expression patterns

  • Detection with domain-specific antibodies matches predicted isoform structure

• Interpretation framework:

  • 522 kDa band: Intact full-length LRP2

  • 280-330 kDa bands: Either proteolytic fragments or established "gp330" form

  • Smaller specific bands: Potential alternative isoforms if reproducible

  • Variable bands: Likely proteolytic artifacts

This analytical approach acknowledges that LRP2 was initially known as gp330 due to its migration at approximately 330 kDa position, representing either a stable proteolytic product or a distinct isoform .

What considerations should guide the interpretation of LRP2 immunostaining patterns in disease states?

Interpreting LRP2 immunostaining patterns in pathological conditions requires systematic analysis:

• Normal vs. pathological pattern comparison:

  • Normal tissues: Strong brush border staining in proximal tubules, weak/segmental podocyte staining

  • Anti-LRP2 nephropathy: Granular deposits in tubular basement membrane, podocytes

  • Other kidney diseases: Variable changes in expression pattern and intensity

• Assessment framework for tubular patterns:

  • Distribution: Proximal vs. distal tubules

  • Subcellular localization: Brush border, cytoplasmic, basolateral

  • Pattern: Linear, granular, or diffuse

  • Intensity: Upregulation or downregulation compared to normal

• Glomerular pattern analysis:

  • Location: Podocytes, mesangium, basement membrane

  • Distribution: Global vs. segmental

  • Colocalization: With immune deposits, complement components

  • Correlation with electron microscopy findings

• Disease-specific interpretation guidelines:

  • Anti-LRP2 nephropathy: Granular TBM and segmental GBM deposits positive for LRP2 and IgG

  • Proteinuric states: Potential redistribution from brush border to cytoplasm

  • Tubular injury: Loss of brush border staining

  • Autoimmune conditions: Colocalization with immune complexes

A critical diagnostic feature of anti-LRP2 nephropathy is the granular tubular basement membrane staining for LRP2, which colocalized with IgG in the immune deposits. This pattern was observed in all ten patients with anti-LRP2 nephropathy but was negative in 40 controls with tubular basement membrane deposits of other causes, demonstrating high sensitivity and specificity .