LRP3 Antibody

What are the key differences between N-terminal and C-terminal LRP3 antibodies?

N-terminal and C-terminal LRP3 antibodies target distinct regions of the LRP3 protein, offering complementary research applications:

N-terminal LRP3 antibodies:

• Target the amino-terminal region of LRP3, such as antibodies recognizing amino acids 17-66

• Particularly useful for detecting full-length LRP3 protein

• Important for studying receptor activation, as this region may contain binding sites for ligands

• Example: ABIN7188855 targets the N-terminal region of human LRP3 and is applicable for Western Blotting and ELISA

C-terminal LRP3 antibodies:

• Target the carboxy-terminal region, such as antibodies recognizing amino acids 661-692

• Valuable for detecting potential proteolytic fragments

• Essential for studying intracellular signaling domains

• Particularly useful for analyzing membrane localization and internalization mechanisms

• Example: RayBiotech's Anti-LRP3 (C-term) recognizes the human LRP3 C-terminal region and is applicable for Western Blotting and IHC-P

For comprehensive studies of LRP3 biology, using both N-terminal and C-terminal antibodies provides complementary data about protein expression, processing, and localization.

How do polyclonal and monoclonal LRP3 antibodies compare in research applications?

When selecting between these antibody types, consider that polyclonal antibodies offer advantages for initial LRP3 characterization and potentially higher sensitivity, while monoclonal antibodies provide superior specificity for targeted epitope analysis and long-term reproducibility in extended studies .

What are the optimal dilutions and protocols for using LRP3 antibodies in Western blotting?

Recommended dilution ranges:

• For C-terminal LRP3 antibodies: 1:500-1:2000

• For N-terminal LRP3 antibodies: 1:500-1:1000

Optimized Western blotting protocol for LRP3 detection:

• Sample preparation:

  • Use membrane-enriched fractions (30 μg) as LRP3 is a membrane protein

  • For cell lysates, NCI-H292 or CHO-PS70 cells provide reliable LRP3 expression

• Gel selection and separation:

  • Use 7.5-12% SDS-PAGE gels or precast 4-15% gradient gels

  • Run samples after boiling at 98°C for 5 min in 6× Laemmli sample buffer

• Transfer and blocking:

  • Transfer proteins to nitrocellulose membranes

  • Block with 5% non-fat milk in TBST

• Antibody incubation:

  • Primary antibody: Incubate at recommended dilution overnight at 4°C

  • Secondary antibody: Use fluorescent secondary antibodies (e.g., IRDye at 1:10000) for quantitative analysis

• Detection:

  • Visualize using chemiluminescence or fluorescent imaging systems like Odyssey CLx

  • Expected molecular weight: ~82-83 kDa (calculated) , though often observed at approximately 110 kDa

For optimal results, include positive controls with verified LRP3 expression and appropriate negative controls to ensure antibody specificity.

How can LRP3 antibodies be effectively used in immunohistochemistry and immunocytochemistry?

Immunohistochemistry (IHC) Optimization:

• Tissue preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used

  • Human skeletal muscle and brain tissue samples provide good LRP3 expression models

• Antigen retrieval:

  • Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or TE buffer (pH 9.0)

  • For neuronal tissues, citrate buffer may preserve morphology better

• Antibody dilutions:

  • For C-terminal antibodies: 1:10-1:50 dilution range for optimal staining

  • For antibodies targeting AA 184-435: Follow manufacturer-specific recommendations (typically 1:100-1:200)

• Detection systems:

  • Use DAB (3,3'-diaminobenzidine) chromogen for brightfield visualization

  • For fluorescent double-labeling with other markers, use appropriate fluorophore-conjugated secondary antibodies

Immunocytochemistry (ICC) Notes:

• Cell models:

  • SH-SY5Y neuroblastoma cells show good endogenous LRP3 expression

  • CHO-PS70 cells transfected with LRP3 exhibit distinct localization patterns in discrete areas of the soma and plasma membrane

• Visualization:

  • LRP3 typically appears as small granules localized in the cytoplasm and proximal dendrites of neurons

  • In glial cells, LRP3 is observed around the nucleus

For both applications, include negative controls (omitting primary antibody) and positive controls (tissues/cells with known LRP3 expression) to validate staining specificity.

What are common issues when detecting LRP3 in Western blotting and how can they be resolved?

Special considerations:

• The calculated molecular weight for LRP3 is 82.9 kDa, but it frequently appears at ~110 kDa due to post-translational modifications

• For more specific detection, use affinity-purified antibodies that underwent epitope-specific immunogen purification

• When studying neuronal tissues, post-mortem delay can significantly impact LRP3 detection; adjust protocols accordingly by increasing antibody concentration for older samples

How can antibody specificity for LRP3 be validated in experimental systems?

Comprehensive LRP3 antibody validation strategies:

• Genetic approaches:

  • siRNA/shRNA knockdown of LRP3 in relevant cell lines (e.g., SH-SY5Y, CHO-PS70)

  • CRISPR/Cas9-mediated knockout of LRP3 (similar to NLRP3 knockout validation systems)

  • Transient overexpression of tagged LRP3 constructs

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide

  • Run parallel Western blots with blocked and unblocked antibody

  • Specific bands should disappear in the blocked condition

• Cross-reactivity assessment:

  • Test antibody against recombinant LRP3 protein

  • Evaluate cross-reactivity with other LDL receptor family members

  • Confirm species reactivity across human, mouse, and rat samples

• Comparative antibody analysis:

  • Use multiple antibodies targeting different LRP3 epitopes (N-terminal vs. C-terminal)

  • Compare staining/binding patterns between antibody sources

  • Consistent detection across antibodies increases confidence in specificity

• Application-specific validations:

  • For Western blotting: Look for a band at the expected molecular weight (~83-110 kDa)

  • For IHC/ICC: Compare staining patterns with published literature showing subcellular distribution in neurons (cytoplasmic granules and proximal dendrites)

  • For immunoprecipitation: Confirm pulled-down protein identity by mass spectrometry

Following these validation strategies ensures experimental results are truly reflective of LRP3 biology rather than antibody artifacts.

How can LRP3 antibodies be used to investigate LRP3's interaction with apoE and APP in Alzheimer's disease models?

LRP3 has been shown to interact with apolipoprotein E (apoE) and amyloid precursor protein (APP), suggesting a role in Alzheimer's disease pathophysiology . Here's a methodological approach to investigate these interactions:

Co-immunoprecipitation (Co-IP) protocol:

• Tissue/cell preparation:

  • Use human frontal cortex extracts from middle-aged individuals and AD-related pathology cases

  • Alternatively, employ CHO-PS70 cells that express wild-type APP770 isoform

• Immunoprecipitation strategy:

  • Incubate membrane-enriched fractions with anti-LRP3 antibody (polyclonal antibodies targeting the C-terminal region work well)

  • Capture antibody-protein complexes using protein A/G magnetic beads

  • Elute bound proteins and analyze by Western blotting

• Detection of interacting partners:

  • Probe membranes with antibodies against:

    • APP (C-terminal or N-terminal antibodies)

    • apoE

    • sAPPα and sAPPβ fragments

• Controls and validation:

  • Perform reverse IP with anti-APP or anti-apoE antibodies

  • Include negative controls (non-specific IgG)

  • Validate with chloroquine treatment to block lysosomal degradation

Fluorescence co-localization analysis:

• Sample preparation:

  • Fix cells/tissue sections with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

• Double immunostaining:

  • Incubate with anti-LRP3 antibody and either anti-APP or anti-apoE antibodies

  • Use differentially labeled secondary antibodies (e.g., Alexa Fluor 488/594)

• Analysis:

  • Capture images using confocal microscopy

  • Quantify co-localization using Pearson's correlation coefficient

  • Compare co-localization patterns between control and AD-model samples

This approach has revealed that LRP3 and APP co-immunoprecipitate in CHO-PS70 cells, and that LRP3 overexpression significantly reduces full-length APP levels, suggesting that LRP3 may influence APP processing through lysosomal degradation/autophagy mechanisms .

What methodologies can be used to study LRP3's role in lysosomal degradation of APP?

Research has demonstrated that LRP3 may regulate APP processing through lysosomal degradation pathways . Here's a detailed methodological approach to investigate this process:

1. Lysosomal inhibition assay:

• Experimental design:

  • Transfect cells (CHO-PS70 or SH-SY5Y) with LRP3 expression constructs

  • Treat cells with chloroquine (10-50 μM) to block lysosomal function for 16-24 hours

  • Analyze APP levels in cell extracts by Western blotting

• Key measurements:

  • Full-length APP levels in membrane fractions

  • APP-CTF fragments in intracellular vesicle fractions

  • sAPPα and sAPPβ levels in culture media

  • Changes in LC3B-I to LC3B-II conversion as autophagy marker

2. Co-localization with lysosomal markers:

• Immunofluorescence approach:

  • Triple staining for LRP3, APP, and lysosomal markers (LAMP1 or LAMP2)

  • Quantitative analysis of co-localization percentages

  • Time-course experiments to track APP trafficking

• Live-cell imaging:

  • Express fluorescently-tagged LRP3 and APP constructs

  • Monitor trafficking and co-localization in real-time

  • Analyze speed and directionality of vesicular movement

3. Mechanistic inhibition studies:

• Targeted approach:

  • Use specific inhibitors of endocytosis (Dynasore)

  • Apply inhibitors of intracellular trafficking (Brefeldin A)

  • Employ targeted siRNAs against autophagy components (ATG5, ATG7)

• Readouts:

  • Measure changes in APP processing products (sAPPα, sAPPβ, Aβ)

  • Quantify APP degradation rates under different conditions

  • Assess LRP3-APP binding affinities with and without inhibitors

Based on existing research, LRP3 overexpression decreases full-length APP levels and APP-CTF in plasma membrane fractions, as well as soluble APP fragments (sAPPα, sAPPβ) and Aβ levels . When lysosomal function is impaired by chloroquine treatment, full-length APP and sAPPα levels increase significantly (p=0.0044 and p=0.031, respectively) compared to non-treated cells , supporting LRP3's role in APP degradation through the lysosomal pathway.

How does LRP3 expression change in Alzheimer's disease progression and how can this be effectively studied with LRP3 antibodies?

Research has shown complex patterns of LRP3 expression across Alzheimer's disease (AD) progression. Here's a methodological approach to study these changes using LRP3 antibodies:

Tissue-based expression analysis:

• Sample collection strategy:

  • Obtain human frontal cortex samples from:

    • Middle-aged control subjects (MA, mean age 51.8±4.8 years)

    • AD-related pathology subjects at different Braak stages:

      • Braak I-II (early, 68.4±8.8 years)

      • Braak III-IV (intermediate, 80.4±8.8 years)

      • Braak V-VI (advanced, 76.5±9.7 years)

• Quantitative expression analysis:

  • Perform quantitative RT-PCR for LRP3 mRNA levels

  • Conduct Western blot analysis using membrane-enriched fractions (30 μg)

  • Use both N-terminal and C-terminal antibodies to detect potential proteolytic fragments

• Spatial analysis through IHC:

  • Perform immunohistochemistry on brain sections

  • Double-label with markers for neurons (NeuN) and glia (GFAP)

  • Quantify LRP3 immunoreactivity across brain regions affected by AD pathology

Challenges and methodological considerations:

• LRP3 immunostaining shows marked individual disparities in both control and AD cases, likely due to vulnerability to pre-mortem status and post-mortem delay

• This variability necessitates larger sample sizes and careful matching of cases for post-mortem interval

• For more reliable quantification, Western blot analysis of membrane-enriched fractions may provide more consistent results than immunohistochemistry-based densitometry

Functional correlation:

• Correlate LRP3 expression levels with:

  • APP processing markers (Aβ load, APP-CTF levels)

  • Cognitive performance scores when available

  • Other LDL receptor family members (especially ApoER2)

While difficult to quantify due to individual variability, immunofluorescence studies have shown that LRP3 antibodies recognize small granules localized in the cytoplasm and proximal dendrites of neurons, and around the nucleus of glial cells in the hippocampus and frontal cortex of human brain tissue .

What methodological approaches can be used to study LRP3's potential therapeutic targeting in neurodegenerative diseases?

Given LRP3's role in APP processing and potential involvement in neurodegenerative pathways, several methodological approaches can be employed to investigate its therapeutic potential:

1. Antibody-based targeting strategies:

• Direct LRP3 targeting:

  • Develop function-blocking antibodies against LRP3's extracellular domain

  • Test their efficacy in cell and animal models

  • Measure changes in APP processing, Aβ production, and cognitive outcomes

• Bispecific antibody approach:

  • Drawing from NLRP3 research methodologies, develop bispecific antibodies targeting LRP3 and a cell-entry receptor

  • Test in cellular models of Alzheimer's disease

  • Analyze changes in APP degradation and Aβ production

2. Mechanistic intervention studies:

• LRP3 overexpression system:

  • Create inducible LRP3 expression systems in neuronal models

  • Measure effects on APP processing and degradation pathways

  • Identify the minimal functional domain required for APP degradation

• Inhibitory approaches:

  • Develop small molecule inhibitors targeting LRP3-APP interaction

  • Use peptide-based competitors of the interaction

  • Test compounds in cellular and animal models of Alzheimer's disease

3. In vivo evaluation methods:

• Viral vector delivery:

  • Use AAV vectors to deliver LRP3 or inhibitory constructs to specific brain regions

  • Apply stereotactic injections in AD mouse models

  • Evaluate effects on local Aβ deposition and neuronal health

• Passive immunotransfer:

  • Similar to anti-LRP/LR antibody W3 approach for prion diseases

  • Deliver anti-LRP3 antibodies through passive immunotransfer

  • Evaluate effects on APP processing and Alzheimer's-like pathology

4. Readout methodologies:

• Histopathological analysis:

  • Immunostaining for Aβ plaques, phospho-tau, and neuroinflammatory markers

  • Quantitative analysis of plaque burden and neuronal loss

• Biochemical assessments:

  • Measure APP, APP fragments, and Aβ levels in brain extracts

  • Analyze LRP3-APP binding under treatment conditions

• Functional evaluation:

  • Cognitive testing in animal models

  • Electrophysiological assessment of synaptic function