LRP1 Antibody, Biotin conjugated

What is LRP1 and what is its significance in neuroscience research?

LRP1 (Low Density Lipoprotein Receptor-Related Protein 1) is a multifunctional cell surface receptor also known as alpha-2-macroglobulin receptor. It has a calculated molecular weight of approximately 505 kDa, though the cleaved extracellular domain is observed at 85-90 kDa in many experimental contexts . LRP1 has gained significant attention in neuroscience research due to its critical role in receptor-mediated transcytosis (RMT) across the blood-brain barrier (BBB) . The expression levels of LRP1 at the BBB correlate well among species, including humans, monkeys, and mice, making it an excellent target for comparative studies . Furthermore, the amino acid sequences of its ligand-binding domains, particularly clusters 2 (CL2) and 4 (CL4), are highly conserved across species, which enhances its utility as a research target . LRP1 is particularly significant for drug delivery system (DDS) development targeting central nervous system (CNS) disorders, as it offers a pathway for therapeutic molecules to cross the typically impermeable BBB .

What applications is the LRP1 antibody (biotin conjugated) suitable for?

The biotin-conjugated LRP1 antibody is suitable for multiple research applications, offering versatility for comprehensive protein analysis:

The biotin conjugation provides enhanced sensitivity through amplification using streptavidin-based detection systems, making it particularly valuable for detecting low-abundance LRP1 in complex biological samples .

What is the specificity of the biotin-conjugated LRP1 antibody?

The biotin-conjugated LRP1 antibody (such as ABIN7471097) specifically recognizes the 85 kDa cleaved fragment of LRP1, corresponding to its extracellular domain . This specificity is crucial for research applications focused on the processed form of the receptor rather than the full-length 505 kDa protein. The antibody has been validated against human and rabbit samples, showing consistent specificity across these species . For the polyclonal antibody variant (such as 26106-1-AP), specificity has been demonstrated in human and mouse samples, with positive Western blot detection in HEK-293 and HeLa cells . It's important to note that different antibodies may target different epitopes within the LRP1 protein, such as specific amino acid regions (e.g., AA 20-270, AA 4441-4544) or terminal regions (C-Term), which should be selected based on the specific research question .

What are the recommended storage conditions for LRP1 antibody?

For optimal performance and longevity of the LRP1 antibody, the following storage conditions are recommended:

Proper storage is essential to maintain antibody integrity and consistent experimental results over time. Repeated freeze-thaw cycles should be minimized as they can lead to degradation of the antibody and reduced performance in applications.

What dilutions are recommended for different applications of LRP1 antibody?

The effective use of LRP1 antibody requires appropriate dilution for each application type:

It is strongly recommended to titrate the antibody in each testing system to obtain optimal results, as the ideal dilution can vary based on sample type, preparation method, and detection system .

How can I optimize LRP1 antibody usage for detecting the cleaved 85 kDa fragment in Western blotting?

Optimizing Western blotting conditions for the 85 kDa cleaved fragment of LRP1 requires attention to several critical parameters:

• Sample preparation: For efficient extraction of membrane-bound LRP1, use lysis buffers containing 1% Triton X-100 or NP-40. Include protease inhibitors to prevent further degradation of the target fragment during preparation.

• Gel selection: Use 8-10% SDS-PAGE gels for optimal resolution of the 85 kDa fragment. Higher percentage gels may not allow proper migration of the protein.

• Transfer conditions: Transfer to PVDF membranes at 100V for 90 minutes in wet transfer systems, or use program settings optimized for high molecular weight proteins in semi-dry systems.

• Blocking optimization: A 5% BSA blocking solution often provides better results than milk-based blockers, especially when using biotin-conjugated antibodies to avoid interference from endogenous biotin in milk.

• Detection system: For biotin-conjugated LRP1 antibodies, use streptavidin-HRP conjugates at 1:5000-1:10000 dilution. Consider enhanced chemiluminescence (ECL) reagents with extended signal duration for reliable detection .

• Exposure time: Start with shorter exposure times (30 seconds) and incrementally increase if needed, as overexposure can lead to background issues with biotin detection systems.

• Control samples: Include positive controls such as HEK-293 or HeLa cell lysates where LRP1 expression has been confirmed .

Researchers should be aware that the 85 kDa fragment represents the cleaved extracellular domain of LRP1, and different sample preparation methods may affect the ratio of cleaved to full-length protein observed in the experiment.

What are the crucial considerations for using LRP1 antibody in blood-brain barrier research?

When employing LRP1 antibodies for blood-brain barrier (BBB) research, consider these critical aspects:

• Model selection:

  • In vitro models: Rat and monkey BBB models have shown validation for LRP1 studies, with 13% and 17% penetration rates respectively for LRP1-binding peptides like KS-487 .

  • Ex vivo models: Brain slices maintain BBB architecture but require rapid processing to preserve integrity.

  • In vivo models: Consider species differences in LRP1 expression, though conservation is high across species.

• Antibody vs. peptide approaches: LRP1-binding peptides such as Angiopep-2 (ANG2) and L57 have been used for studying BBB penetration, with newer options like KS-487 showing promising results (EC₅₀ value of 10.5 nM for binding to LRP1 Cluster 4 domain) . Consider whether antibody detection or peptide-based functional studies better serve your research question.

• Permeability assessment:

  • When evaluating BBB penetration, standardize incubation times (e.g., 24h was used in KS-487 studies) .

  • Consider dual labeling approaches using biotin-conjugated antibodies with fluorescent secondary detection.

• Specificity controls: Include competition assays with known LRP1 ligands to verify specificity of antibody binding at the BBB.

• Translational relevance: Note that LRP1 expression at the BBB correlates well among species, making findings potentially more translatable to human applications .

For researchers developing drug delivery systems targeting the CNS, LRP1 antibodies can serve both as detection tools and as potential targeting moieties, though peptides like KS-487 may offer advantages in terms of size and BBB penetration efficiency .

How can I develop a competitive binding assay to evaluate LRP1-binding activity using biotin-conjugated antibodies?

A competitive binding assay for LRP1 can be designed based on the principles described for peptide binding studies , with modifications for antibody-based detection:

• Assay principle: The basic approach involves competition between biotinylated and non-biotinylated molecules for binding to LRP1, particularly to its Cluster 4 (CL4) domain.

• Materials required:

  • Purified recombinant LRP1(CL4) domain

  • Biotin-conjugated LRP1 antibody (e.g., ABIN7471097)

  • Non-biotinylated competitor (test molecule)

  • Streptavidin-HRP or streptavidin-fluorophore

  • Appropriate detection system (colorimetric, fluorescent, or luminescent)

• Protocol outline:

  • Immobilize LRP1(CL4) on a solid phase (e.g., 96-well plate)

  • Pre-incubate varying concentrations of non-biotinylated competitor with a fixed concentration of biotin-conjugated LRP1 antibody

  • Add the mixture to the immobilized LRP1(CL4)

  • After washing, detect bound biotin-conjugated antibody using streptavidin-based detection

• Controls and validation:

  • Include a concentration series of biotin-conjugated antibody alone to establish a standard curve

  • Use known LRP1 ligands as positive controls (e.g., Angiopep-2, L57)

  • Include a non-binding antibody as a negative control

• Data analysis:

  • Calculate percent inhibition at each competitor concentration

  • Determine IC₅₀ values for each competitor

  • Compare binding affinities across different molecules

This approach can be extended to evaluate stability in plasma by incubating the competitor molecules in plasma before the competition assay, similar to the method used for KS-487 peptide evaluation . Decreased competitive inhibition after plasma incubation indicates degradation of the test molecule.

What methods are recommended for validating LRP1 antibody specificity in different experimental systems?

Comprehensive validation of LRP1 antibody specificity should incorporate multiple complementary approaches:

• Genetic validation:

  • Use LRP1 knockout or knockdown models (cells or tissues)

  • Compare antibody staining/signal between wild-type and KO/KD samples

  • Expected outcome: Loss or significant reduction of signal in KO/KD samples

• Peptide competition:

  • Pre-incubate antibody with excess immunizing peptide

  • Apply the mixture to your experimental system

  • Expected outcome: Significant reduction in specific signal

• Orthogonal detection methods:

  • Compare results across multiple techniques (WB, IHC, IF, FACS)

  • Use antibodies targeting different epitopes of LRP1

  • Expected outcome: Consistent detection pattern across methods

• Mass spectrometry validation:

  • Perform immunoprecipitation using the LRP1 antibody

  • Analyze pulled-down proteins by mass spectrometry

  • Expected outcome: Identification of LRP1 peptides as major components

• Cross-species reactivity:

  • Test antibody performance across stated reactive species

  • For the biotin-conjugated LRP1 antibody, verify reactivity in both human and rabbit samples

  • For polyclonal variants, check human and mouse reactivity

  • Expected outcome: Consistent detection at appropriate molecular weight

• Fragment-specific validation (particularly important for LRP1):

  • Verify detection of the expected 85 kDa cleaved fragment in Western blotting

  • Confirm specificity for extracellular domain

  • Expected outcome: Primary detection at the stated molecular weight

Documentation of these validation steps significantly enhances the reliability and reproducibility of research findings involving LRP1 antibodies.

How do I troubleshoot weak or non-specific staining when using LRP1 antibodies in immunohistochemistry of brain tissue?

When encountering issues with LRP1 immunohistochemistry in brain tissue, systematically address these potential problems:

For LRP1 detection specifically, note that the antibody recognizes the 85 kDa cleaved fragment (extracellular domain) , which may have different accessibility in various fixation and tissue preparation methods. If troubleshooting the biotin-conjugated antibody, consider testing unconjugated versions to determine if the biotin modification affects tissue penetration or epitope recognition .

How can I compare the efficiency of LRP1-binding peptides versus antibodies for blood-brain barrier studies?

Comparing LRP1-binding peptides and antibodies requires evaluation across multiple parameters:

Experimental design considerations:

• For comparative studies, label both peptides and antibodies with equivalent detection tags (fluorescent dyes, radioisotopes).

• When assessing BBB penetration:

  • Use consistent incubation times (24h has been validated in rat and monkey BBB models)

  • Compare molar equivalent amounts rather than mass-based dosing

  • Include positive controls (e.g., Angiopep-2 peptide)

• For binding competition studies:

  • Determine whether peptides and antibodies bind to the same or different epitopes on LRP1

  • Establish whether binding is mutually exclusive or cooperative

The choice between peptides and antibodies often depends on the specific research goal. Peptides like KS-487 may be preferred for drug delivery applications due to their smaller size and documented BBB penetration , while antibodies excel in detection applications where sensitivity and specificity are paramount .

What are the optimized protocols for using biotin-conjugated LRP1 antibodies in multi-color immunofluorescence experiments?

For successful multi-color immunofluorescence using biotin-conjugated LRP1 antibodies, follow this optimized protocol:

Materials:

• Biotin-conjugated LRP1 antibody (e.g., ABIN7471097)

• Streptavidin conjugated to compatible fluorophore (e.g., Streptavidin-Alexa Fluor 488)

• Additional primary antibodies from different host species

• Species-specific secondary antibodies with non-overlapping fluorophores

• DAPI for nuclear counterstaining

• Mounting medium with anti-fade properties

Protocol:

• Tissue/Cell Preparation:

  • Fix samples with 4% paraformaldehyde (10-15 minutes for cells, 24h for tissues)

  • For brain tissue, perform antigen retrieval with TE buffer pH 9.0

  • Permeabilize with 0.1-0.3% Triton X-100 (if detecting intracellular epitopes)

• Blocking:

  • Block with 5-10% normal serum (from the same species as secondary antibodies)

  • Critical step: Include streptavidin/biotin blocking kit to eliminate endogenous biotin signal

  • Block for 1-2 hours at room temperature

• Primary Antibody Incubation:

  • Apply biotin-conjugated LRP1 antibody at 1:100-1:500 dilution

  • Simultaneously apply other primary antibodies from different host species

  • Incubate overnight at 4°C

• Washing:

  • Wash 3x15 minutes with PBS containing 0.1% Tween-20

• Secondary Detection:

  • Apply species-specific secondary antibodies for non-biotinylated primaries

  • Apply streptavidin-fluorophore conjugate (1:1000-1:5000)

  • Incubate 1-2 hours at room temperature in the dark

• Final Washing and Mounting:

  • Wash 3x15 minutes with PBS containing 0.1% Tween-20

  • Counterstain with DAPI (1:10000) for 5-10 minutes

  • Mount with anti-fade medium

Critical Considerations:

• Perform controls omitting primary antibodies to assess background

• Include single-color controls for spectral compensation

• When using multiple rabbit antibodies, consider sequential immunostaining with careful stripping between rounds

• For co-localization studies with other LRP1 domain-specific antibodies, check antibody compatibility to ensure non-overlapping epitopes

This protocol is optimized for detecting the 85 kDa cleaved extracellular domain of LRP1, which the biotin-conjugated antibody specifically recognizes .

How can I integrate LRP1 antibody detection with functional studies of receptor-mediated transcytosis?

Integrating structural detection with functional analysis of LRP1-mediated transcytosis requires a multi-faceted approach:

• Dual-purpose experimental design:

  • Combine antibody-based detection with cargo transport assays

  • Use different compartments in transwell systems to assess directional transport

• Quantitative co-localization analysis:

  • Label LRP1 with biotin-conjugated antibody and fluorescent streptavidin

  • Label cargo molecules with a spectrally distinct fluorophore

  • Analyze co-localization during transport using:

    • Pearson's correlation coefficient

    • Mander's overlap coefficient

    • Object-based co-localization analysis

• Live cell imaging approaches:

  • Use Fab fragments derived from LRP1 antibodies for reduced interference

  • Combine with pH-sensitive cargo labels to track endosomal processing

  • Implement pulse-chase labeling protocols to follow trafficking kinetics

• Functional integration strategies:

  • Compare antibody-based detection with peptide-based functional studies

  • Correlate LRP1 expression levels with transport efficiency of model substrates

  • Use competition assays with LRP1-binding peptides like KS-487, Angiopep-2, or L57

• Validation in multiple model systems:

  • In vitro: Use established BBB models validated for LRP1 studies (rat and monkey models have shown 13% and 17% penetration rates respectively)

  • Ex vivo: Brain slice cultures maintain cytoarchitecture while allowing controlled access

  • In vivo: Consider intravital imaging approaches with minimally invasive LRP1 labeling

• Comparative assessment protocol:

  • Establish baseline transport rates of model cargos (e.g., α2-macroglobulin)

  • Assess changes when blocking LRP1 with antibodies or competing peptides

  • Quantify correlation between LRP1 expression (by antibody labeling) and transport efficiency

This integrated approach enables researchers to connect structural information about LRP1 distribution and expression with functional data on its role in transcytosis, providing a more complete understanding of BBB transport mechanisms.

What are the emerging trends in LRP1 antibody applications for neurodegenerative disease research?

LRP1 antibodies are increasingly being utilized in neurodegenerative disease research across several innovative directions. The development of novel LRP1-binding peptides such as KS-487 represents a significant advancement in blood-brain barrier penetration strategies, with documented efficiency in both rat and monkey BBB models . These peptides, with binding affinities in the nanomolar range (EC₅₀ = 10.5 nM), are complementing traditional antibody approaches by offering new tools for drug delivery to the CNS .

The biotin conjugation of LRP1 antibodies enhances detection sensitivity through signal amplification systems, which is particularly valuable for studying LRP1's role in complex neurodegenerative pathologies where protein expression may be altered . Furthermore, the multi-application capability of these antibodies—spanning Western blotting, immunohistochemistry, immunofluorescence, flow cytometry, and immunoprecipitation—enables comprehensive investigation of LRP1 biology from molecular interactions to cellular distribution .

As research continues to elucidate LRP1's roles beyond simple cargo transport to include signaling functions and interactions with neurodegenerative disease-associated proteins, antibody-based approaches will remain essential tools for mechanistic studies. The integration of antibody detection with functional peptide studies represents a particularly promising direction for translational research targeting the blood-brain barrier in conditions such as Alzheimer's disease, Parkinson's disease, and other neurological disorders.

What quality control measures should be implemented when working with LRP1 antibodies across different experimental platforms?

Implementing rigorous quality control measures when working with LRP1 antibodies ensures reliable and reproducible research outcomes:

• Initial validation requirements:

  • Confirm antibody specificity through genetic validation models (knockout/knockdown)

  • Verify detection of the appropriate molecular weight forms (505 kDa full-length protein vs. 85 kDa cleaved fragment)

  • Document cross-reactivity with intended species (human, mouse, rabbit as applicable)

• Application-specific validation:

  • For each new application, tissue type, or sample preparation method, include positive and negative controls

  • Document optimized dilution factors for each application (e.g., 1:500-1:2000 for WB, 1:50-1:500 for IHC)

  • Maintain detailed records of lot-to-lot variation in performance

• Regular quality checks:

  • Periodically test antibody performance against reference samples

  • Monitor for changes in signal intensity or background over time

  • Implement stability testing protocols for long-term storage

• Standardized reporting:

  • Document key antibody metadata (catalog number, lot, host, clonality, epitope)

  • Include comprehensive methods sections in publications

  • Consider following antibody reporting guidelines such as those recommended by scientific journals

• Cross-validation strategy:

  • Use multiple antibodies targeting different epitopes of LRP1

  • Compare biotin-conjugated antibodies with unconjugated versions

  • Validate findings with orthogonal detection methods