LRPAP1 Antibody, HRP conjugated

What is LRPAP1 and what is its functional significance in cellular biology?

LRPAP1 functions as a molecular chaperone for LDL receptor-related proteins that regulates their ligand binding activity along the secretory pathway . It is also known by several alternative names including A2MRAP, Alpha-2-macroglobulin receptor-associated protein, Alpha-2-MRAP, and RAP . The protein plays a critical role in the proper folding and trafficking of LDL receptor family members, preventing premature ligand binding during synthesis and transport.

Beyond its chaperone function, LRPAP1 has been identified as an autoantigenic B-cell receptor (BCR) target in approximately one-third of all Mantle Cell Lymphomas (MCLs), suggesting its significant role in lymphoma pathogenesis . This discovery has expanded our understanding of LRPAP1 beyond its conventional role in lipoprotein receptor biology and highlighted its potential importance in immune system dysfunction and cancer development.

What experimental applications are LRPAP1 antibodies suitable for in laboratory research?

LRPAP1 antibodies have demonstrated utility across multiple laboratory applications, making them versatile tools for research. They are suitable for several techniques including:

• Immunohistochemistry on paraffin-embedded tissues (IHC-P)

• Immunoprecipitation (IP)

• Western blotting (WB)

• Flow cytometry (particularly for detecting LRPAP1-reactive B-cell receptors)

For Western blot applications specifically, optimal dilution ratios have been established at approximately 1:6000 for commercially available LRPAP1 antibodies, though this may vary between manufacturers and specific experimental conditions . The antibodies have been validated across multiple species including human, mouse, and rat samples, making them suitable for comparative studies across model organisms . When conjugated with HRP, these antibodies can offer direct detection without the need for secondary antibodies, streamlining experimental protocols while maintaining sensitivity.

What is the expected molecular weight of LRPAP1 in Western blot applications?

In Western blot applications, researchers should be aware of an important discrepancy between the predicted and observed molecular weights of LRPAP1. While the predicted molecular weight based on amino acid composition is approximately 41 kDa, the observed band size in Western blot experiments is typically around 45 kDa . This difference of approximately 4 kDa is consistently observed across various tissue samples including human, mouse, and rat kidney tissues, as well as in cell lines such as 293T cells .

This discrepancy between predicted and observed molecular weight is likely due to post-translational modifications of the protein, such as glycosylation or phosphorylation. Understanding this difference is crucial for accurate interpretation of Western blot results, as identifying the correct band is essential for avoiding false positives or negatives. When using HRP-conjugated LRPAP1 antibodies, this size difference remains consistent and can serve as an important validation parameter for confirming specific detection.

How should blocking conditions be optimized for LRPAP1 antibody detection?

Optimizing blocking conditions is crucial for achieving specific detection of LRPAP1 while minimizing background signal, particularly when using HRP-conjugated antibodies where direct enzymatic activity amplifies both specific and non-specific signals. Based on established protocols, the following blocking conditions have proven effective:

• Blocking buffer composition: 5% non-fat dry milk (NFDM) in TBST has demonstrated optimal results for minimizing background while preserving specific LRPAP1 detection

• Blocking duration: Overnight incubation at 4°C provides superior background reduction compared to shorter blocking periods at room temperature

• Diluting buffer for antibody: 5% NFDM/TBST maintains the same composition as the blocking buffer to ensure consistent protein environment during antibody binding

• Washing steps: A minimum of three thorough washes with TBST between antibody incubations helps remove unbound antibody

When working with HRP-conjugated antibodies specifically, these optimized blocking conditions become even more critical as they help prevent non-specific binding of the conjugated enzyme to the membrane, which could otherwise result in false positive signals during development. The proper balance between sufficient blocking and maintaining antibody accessibility to the target epitope is essential for generating clean, interpretable results.

What tissue and cell types are recommended for studying LRPAP1 expression?

LRPAP1 expression has been successfully studied in various tissue and cell types, providing researchers with several options for positive controls and experimental systems. The following tissues and cell lines have demonstrated reliable LRPAP1 expression:

• Kidney tissues (human, mouse, and rat) have shown consistent LRPAP1 expression across species, making them valuable positive controls for antibody validation

• HEK 293T cells express detectable levels of LRPAP1 and can serve as accessible positive controls in cell culture experiments

• B-cell lymphoma cell lines with differential LRPAP1 reactivity provide important comparative systems:

  • MAVER1 and Z138 (MCL cell lines with LRPAP1-reactive BCRs)

  • Granta-519 and Mino (MCL cell lines without LRPAP1 reactivity)

  • U2932 (DLBCL cell line) has also shown LRPAP1 reactivity

This diversity of expression systems allows researchers to select appropriate positive and negative controls based on their specific experimental goals and available resources. Additionally, the different BCR reactivity profiles of these cell lines make them particularly valuable for studying LRPAP1's role in B-cell malignancies and for validating the specificity of LRPAP1 antibodies in functional studies.

How can researchers differentiate between non-specific binding and true LRPAP1 signal in Western blot applications?

Differentiating between specific LRPAP1 signal and non-specific binding requires a systematic approach, particularly when using HRP-conjugated antibodies where signal amplification can exacerbate background issues:

• Band size verification: True LRPAP1 signal should appear at approximately 45 kDa, despite the predicted molecular weight of 41 kDa . This consistent discrepancy serves as a useful verification parameter, as non-specific binding often presents at unexpected molecular weights.

• Control samples: Include known LRPAP1-expressing tissues such as kidney lysates as positive controls, and cell lines without LRPAP1 expression as negative controls. The contrast between these samples helps confirm signal specificity.

• Antibody titration: Perform serial dilutions of the LRPAP1 antibody (typically from 1:1000 to 1:10,000) to identify the optimal concentration where specific signal remains strong while background is minimized. For HRP-conjugated antibodies, this optimization is particularly critical.

• Blocking optimization: Systematic testing of different blocking agents (NFDM, BSA, casein) can help identify conditions that minimize non-specific binding while preserving true LRPAP1 detection. The established protocol using 5% NFDM/TBST offers a reliable starting point .

• Membrane handling: Careful handling of the membrane, including thorough washing steps and ensuring complete coverage during antibody incubation, helps eliminate technical sources of non-specific binding.

For HRP-conjugated antibodies specifically, include an enzyme activity control (substrate only) to distinguish between enzymatic artifacts and true LRPAP1 detection. These systematic approaches help ensure that observed signals truly represent LRPAP1 rather than technical artifacts or cross-reactivity.

What experimental design considerations are important when using LRPAP1 antibodies in mantle cell lymphoma research?

When designing experiments using LRPAP1 antibodies for mantle cell lymphoma research, several critical factors should be considered:

These design considerations enable researchers to effectively leverage LRPAP1 antibodies for understanding MCL pathogenesis and developing potential therapeutic approaches based on the unique role of LRPAP1 as an autoantigen in this malignancy.

How does HRP conjugation affect the sensitivity and specificity of LRPAP1 antibody detection compared to other detection methods?

HRP conjugation significantly influences LRPAP1 antibody performance in several important ways compared to other detection methods:

Sensitivity considerations:

Specificity considerations:

• Elimination of secondary antibody cross-reactivity represents a major advantage of direct HRP conjugation, reducing background and false positive results.

• The conjugation process may potentially mask or alter certain epitopes, necessitating validation against unconjugated versions of the same antibody.

• HRP-conjugated antibodies eliminate species cross-reactivity issues that can occur with secondary antibodies in multi-labeling experiments.

Comparative detection approaches:

The choice between these approaches should be guided by specific experimental requirements, balancing sensitivity needs against workflow considerations and the importance of multiplexing capabilities.

What troubleshooting approaches should be applied when LRPAP1 antibody staining patterns differ from expected results?

When LRPAP1 antibody staining patterns deviate from expected results, a systematic troubleshooting approach should be employed:

• Signal absence or weak signal:

  • Verify protein transfer efficiency with reversible membrane staining (Ponceau S)

  • Increase antibody concentration incrementally (starting from 1:6000 dilution as reference)

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

  • For tissue sections, test alternative epitope retrieval methods

  • For HRP-conjugated antibodies specifically, verify enzymatic activity with control substrates

• Multiple unexpected bands or high background:

  • Evaluate sample integrity through freshly prepared lysates

  • Increase blocking stringency (longer blocking time or higher concentration of blocking protein)

  • Test more stringent washing conditions (additional washes or increased detergent concentration)

  • Adjust blocking reagent composition (switch from milk to BSA if lipid binding is suspected)

  • For HRP-conjugated antibodies, add hydrogen peroxide quenching step to eliminate endogenous peroxidase activity

• Inconsistent results between replicates:

  • Standardize lysate preparation protocol (consistent lysis buffer, protease inhibitors)

  • Ensure uniform protein loading (verify with total protein staining)

  • Control incubation temperature precisely throughout protocol

  • Prepare fresh working solutions for each experiment

  • Establish positive control standards (kidney tissue lysate) for normalization

• Discrepancy between observed and expected molecular weight:

  • Remember that LRPAP1 typically runs at 45 kDa despite a predicted molecular weight of 41 kDa

  • Consider post-translational modifications that may alter migration

  • Compare with recombinant LRPAP1 standard if available

  • Verify specificity through immunoprecipitation followed by mass spectrometry

For HRP-conjugated antibodies specifically, additional troubleshooting may include substrate optimization and careful timing of development steps to avoid signal saturation while maintaining sufficient sensitivity for detection.

How can LRPAP1 antibodies be incorporated into multiplex immunoassays for studying receptor-related protein pathways?

Incorporating LRPAP1 antibodies into multiplex immunoassays requires careful consideration of several technical aspects:

• Antibody compatibility planning:

  • Select LRPAP1 antibodies raised in different host species than other target antibodies to facilitate discrimination

  • Verify absence of cross-reactivity between LRPAP1 antibody and other primary/secondary antibodies in the multiplex panel

  • Test sequential versus simultaneous antibody application protocols to determine optimal staining approach

• Signal discrimination strategies:

  • For fluorescence-based multiplex assays, select fluorophores with minimal spectral overlap when using fluorophore-conjugated LRPAP1 antibodies

  • For HRP-based multiplex approaches, consider:
    a) Sequential detection with HRP inactivation between rounds
    b) Tyramide signal amplification with different fluorophores
    c) Chromogenic multiplex IHC with distinct precipitates for each target

• Optimization protocol development:

  • Establish single-plex protocols for each target before combining into multiplex format

  • Determine optimal concentration for LRPAP1 antibody in multiplex context, which may differ from single-plex conditions

  • Validate staining patterns in well-characterized control samples expressing known levels of LRPAP1

• Technical considerations for specific applications:

  • For IHC/IF: Test antigen retrieval methods compatible with all targets in the multiplex panel

  • For flow cytometry: Carefully titrate each antibody to avoid compensation challenges

  • For Western blot multiplexing: Consider size separation of targets or sequential stripping and reprobing

• Data analysis approaches:

  • Implement appropriate controls for signal normalization across targets

  • Apply spectral unmixing algorithms for fluorescence applications

  • Develop quantification methods that account for potential signal interactions

When using HRP-conjugated LRPAP1 antibodies specifically in multiplex settings, researchers must carefully plan detection sequence and ensure complete inactivation between detection cycles to prevent signal carryover and false co-localization artifacts.

What is the optimal Western blot protocol for detecting LRPAP1 using HRP-conjugated antibodies?

The following protocol has been optimized for detecting LRPAP1 using HRP-conjugated antibodies in Western blotting applications:

Sample preparation:

• Extract proteins from tissues/cells using RIPA buffer supplemented with protease inhibitors

• Determine protein concentration through Bradford or BCA assay

• Prepare samples at 10-20 μg total protein per lane (10 μg has been validated for kidney tissue lysates and cell lines)

• Denature samples in Laemmli buffer at 95°C for 5 minutes

Gel electrophoresis and transfer:

• Separate proteins on 10% SDS-PAGE gel (optimal for resolving LRPAP1's 45 kDa band)

• Transfer to PVDF membrane using semi-dry transfer system (PVDF has shown superior results for LRPAP1 detection compared to nitrocellulose)

• Verify transfer efficiency with reversible Ponceau S staining

Immunodetection with HRP-conjugated LRPAP1 antibody:

• Block membrane with 5% NFDM/TBST for 1 hour at room temperature or overnight at 4°C

• Incubate with HRP-conjugated LRPAP1 antibody at 1:5000-1:6000 dilution in 5% NFDM/TBST for 2 hours at room temperature

• Wash 3 times, 5 minutes each with TBST

• Develop using chemiluminescence substrate appropriate for HRP detection

• Expose to X-ray film or image using digital imaging system

Controls and validation:

• Include positive control (kidney tissue lysate from human, mouse, or rat)

• Include negative control (non-expressing cell line)

• Expected band size: 45 kDa (though predicted molecular weight is 41 kDa)

• For quantitative analysis, normalize to appropriate loading control

This protocol has been validated across human, mouse, and rat samples, with consistent results across species . The direct HRP conjugation eliminates secondary antibody incubation steps, reducing protocol time and potential sources of background.

How should researchers optimize LRPAP1 antibody conditions for studying mantle cell lymphoma using flow cytometry?

Optimizing LRPAP1 antibody conditions for flow cytometry analysis of mantle cell lymphoma requires careful attention to several key parameters:

• Sample preparation considerations:

  • Cell dissociation: Use enzyme-free dissociation buffers to preserve surface epitopes, particularly for detecting BCR-bound LRPAP1

  • Fixation protocol: 2-4% paraformaldehyde for 10-15 minutes at room temperature provides optimal epitope preservation

  • Permeabilization (for intracellular LRPAP1): 0.1% saponin maintains better morphology while allowing antibody access

• Antibody titration and validation:

  • Perform titration experiments using serial antibody dilutions (typically 1:50 to 1:500)

  • Test antibody performance on known positive controls (MAVER1, Z138) and negative controls (Granta-519, Mino)

  • Determine optimal signal-to-background ratio by comparing specific binding to isotype control

  • For HRP-conjugated antibodies, develop signal using fluorescent tyramide substrates for flow cytometry applications

• Essential controls for interpretation:

  • Unstained cells to establish autofluorescence baseline

  • Isotype control matched to LRPAP1 antibody to assess non-specific binding

  • FMO (Fluorescence Minus One) controls for multi-parameter panels

  • Positive control cell lines with known LRPAP1-reactive BCRs

  • Negative control cell lines with non-LRPAP1-reactive BCRs

• Gating strategy development:

  • Identify viable cells using appropriate viability dye

  • Remove doublets and debris through FSC/SSC gating

  • For B-cell identification, include CD19 or CD20 markers

  • Establish positive/negative cutoffs using control cell lines

• Assay validation approaches:

  • Compare results from multiple antibody clones targeting different LRPAP1 epitopes

  • Correlate flow cytometry results with other detection methods (Western blot, IHC)

  • Assess assay reproducibility through repeated analysis of reference standards

This optimized approach enables reliable detection of LRPAP1-reactive B-cells in both research settings and potential clinical applications for mantle cell lymphoma characterization and monitoring.

What validation steps are essential for confirming LRPAP1 antibody specificity before conducting advanced research?

Validating LRPAP1 antibody specificity is essential for reliable research outcomes. The following comprehensive validation approach is recommended before proceeding with advanced applications:

• Western blot validation:

  • Confirm single band at the expected molecular weight (45 kDa for LRPAP1)

  • Test across multiple cell types/tissues with known differential expression

  • Compare results with alternative LRPAP1 antibodies targeting different epitopes

  • For HRP-conjugated antibodies, compare directly with unconjugated versions to ensure conjugation hasn't altered specificity

• Genetic validation approaches:

  • Test antibody in LRPAP1 knockdown or knockout models

  • Verify diminished or absent signal following genetic manipulation

  • Compare results with mRNA expression levels to confirm correlation

• Immunoprecipitation studies:

  • Perform IP followed by Western blot detection to confirm target recognition

  • For advanced validation, conduct IP followed by mass spectrometry to confirm LRPAP1 identity

  • Verify absence of significant off-target binding

• Competitive binding assays:

  • Pre-incubate antibody with purified LRPAP1 protein

  • Demonstrate reduced or eliminated binding following pre-absorption

  • Include non-relevant protein controls to confirm specificity of competition

• Cross-reactivity assessment:

  • Test across multiple species if cross-reactivity is claimed (human, mouse, rat)

  • Evaluate potential cross-reactivity with structurally related proteins

  • For applications in complex samples, assess performance in the presence of potential interfering substances

This systematic validation ensures that experimental findings reflect genuine LRPAP1 biology rather than artifacts of non-specific binding or cross-reactivity, establishing a solid foundation for subsequent advanced research applications.

What are the critical parameters for successfully using LRPAP1 antibodies in immunoprecipitation studies?

Successful immunoprecipitation (IP) using LRPAP1 antibodies depends on optimizing several critical parameters:

• Lysis buffer optimization:

  • Use NP-40 or RIPA buffer with 1% detergent for balanced solubilization while preserving interactions

  • Include protease inhibitors to prevent LRPAP1 degradation during preparation

  • Add phosphatase inhibitors if studying phosphorylation states of LRPAP1

  • Adjust salt concentration (150-300 mM NaCl) to balance extraction efficiency with preservation of interactions

• Antibody selection and binding conditions:

  • Choose antibodies validated specifically for IP applications

  • Determine optimal antibody-to-lysate ratio through titration experiments

  • Optimize binding conditions:
    a) 4°C overnight incubation often yields best results
    b) Gentle rotation to maintain continuous mixing without foam formation
    c) Pre-clearing lysate with beads alone can reduce non-specific binding

• Bead selection and handling:

  • Protein A/G magnetic beads typically provide efficient capture with low background

  • For HRP-conjugated antibodies, specialized capture systems may be needed

  • Determine optimal bead volume (typically 20-50 μL of slurry per reaction)

  • Implement gentle washing procedures to maintain complex integrity while removing contaminants

• Elution and detection optimization:

  • For Western blot analysis following IP:
    a) Elute in denaturing buffer at 95°C for 5 minutes
    b) Load appropriate IP fraction alongside input control
    c) Expect LRPAP1 detection at approximately 45 kDa

  • For co-IP studies:
    a) Use gentler elution conditions if studying protein interactions
    b) Consider crosslinking antibody to beads to avoid antibody contamination in eluate

• Controls for interpretation:

  • Input control (5-10% of starting material)

  • Negative control using non-specific antibody of same isotype

  • Bead-only control to assess non-specific binding to matrix

  • If available, LRPAP1-depleted lysate as negative control

By optimizing these parameters, researchers can achieve high specificity and sensitivity in LRPAP1 immunoprecipitation, enabling detailed studies of LRPAP1 interactions, post-translational modifications, and complex formation in various biological contexts.

How can researchers optimize immunofluorescence protocols for detecting LRPAP1 in tissue sections?

Optimizing immunofluorescence protocols for LRPAP1 detection in tissue sections requires careful attention to several key aspects:

• Tissue preservation and fixation:

  • Fresh frozen sections: Provide superior epitope preservation but poorer morphology

  • FFPE sections: Better morphology but require optimized antigen retrieval

  • Optimal fixation: 4% paraformaldehyde for 24-48 hours provides good balance between preservation and antibody accessibility

  • Section thickness: 4-6 μm sections offer optimal balance between signal strength and resolution

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval:
    a) Citrate buffer (pH 6.0): Standard starting point for LRPAP1 detection
    b) EDTA buffer (pH 9.0): Alternative for certain LRPAP1 epitopes
    c) Optimization of heating time (typically 15-25 minutes)

  • Enzymatic retrieval alternatives:
    a) Proteinase K treatment: Test at concentrations of 5-20 μg/mL
    b) Trypsin digestion: 0.05-0.1% for 5-15 minutes at 37°C

• Blocking and permeabilization:

  • Blocking buffer composition: 5-10% normal serum (species of secondary antibody) with 0.1-0.3% Triton X-100

  • Blocking duration: 1-2 hours at room temperature

  • Permeabilization: 0.1-0.3% Triton X-100 or 0.1% saponin (gentler alternative)

  • Autofluorescence reduction: Treat sections with 0.1-1% sodium borohydride or commercial autofluorescence quenchers

• Antibody incubation parameters:

  • Primary antibody dilution: Begin testing at 1:100-1:500 range

  • Incubation conditions: Overnight at 4°C in humidified chamber

  • Washing protocol: 3-5 washes with PBS-T, 5-10 minutes each

  • Secondary antibody selection: High-quality fluorophore-conjugated antibodies with minimal cross-reactivity

  • Signal amplification alternatives:
    a) Tyramide signal amplification for HRP-conjugated antibodies
    b) Biotinylated secondary with fluorescent streptavidin

• Controls and counterstaining:

  • Positive control tissue: Kidney sections show reliable LRPAP1 expression

  • Negative control: Primary antibody omission

  • Nuclear counterstain: DAPI (1 μg/mL) or Hoechst 33342

  • Reference markers: Include cell type-specific markers to contextualize LRPAP1 localization

These optimized parameters enable sensitive and specific detection of LRPAP1 in tissue sections while maintaining morphological context, facilitating studies of LRPAP1 expression patterns in normal and pathological tissues.

How can LRPAP1 antibodies be used to investigate the B-cell receptor signaling pathway in lymphoma?

LRPAP1 antibodies offer unique opportunities for investigating B-cell receptor (BCR) signaling in lymphoma, particularly given LRPAP1's role as an autoantigen in mantle cell lymphoma:

• Characterization of LRPAP1-reactive BCR subsets:

  • Identify lymphoma cases with LRPAP1-reactive BCRs using flow cytometry or tissue-based approaches

  • Correlate LRPAP1 reactivity with other molecular and clinical features

  • Estimate frequency of LRPAP1-reactive BCRs across different B-cell malignancy subtypes (approximately one-third of MCL cases)

• Analysis of BCR signaling cascade activation:

  • Use LRPAP1 as a specific stimulus to trigger BCR signaling in responsive lymphoma cells

  • Monitor proximal signaling events:
    a) Calcium flux measurement using fluorescent indicators
    b) Phosphorylation of key signaling molecules (SYK, BTK, PLCγ2)
    c) Activation of downstream transcription factors (NF-κB, NFAT)

  • Compare signaling patterns between LRPAP1-reactive and non-reactive MCL cells

• Correlation with BCR pathway inhibitor sensitivity:

  • Investigate whether LRPAP1-reactive lymphomas show differential sensitivity to BTK inhibitors

  • Determine if LRPAP1 binding alters response to BCR pathway inhibitors

  • Identify potential biomarkers for therapy selection based on LRPAP1 reactivity status

• Probing chronic antigenic stimulation mechanisms:

  • Study how continuous LRPAP1 exposure affects BCR signaling dynamics

  • Investigate potential role in promoting lymphoma cell survival and proliferation

  • Examine changes in BCR internalization and recycling following LRPAP1 binding

• Therapeutic targeting approaches:

  • Develop and test LRPAP1-based strategies like bispecific constructs (anti-CD3/LRPAP1 or anti-CD16/LRPAP1) to redirect immune effector cells

  • Evaluate IgG1-format LRPAP1 BAR bodies for targeting LRPAP1-reactive lymphoma cells

  • Assess cytotoxic effects of these approaches on lymphoma cells with LRPAP1-reactive BCRs

These research applications leverage the specific interaction between LRPAP1 and BCRs in certain lymphoma subtypes, providing insights into both fundamental disease mechanisms and potential therapeutic approaches.

What methods are recommended for using LRPAP1 antibodies to monitor treatment response in lymphoma models?

LRPAP1 antibodies can be effectively employed to monitor treatment response in lymphoma models through several methodological approaches:

• Flow cytometry for minimal residual disease detection:

  • Develop multi-parameter panel including LRPAP1 reactivity assessment

  • Establish detection threshold using dilution experiments with known LRPAP1-reactive cells

  • Protocol optimization:
    a) Use viability dye to exclude dead cells
    b) Include CD19/CD20 for B-cell identification
    c) Analyze LRPAP1-reactive BCR frequency within B-cell population

  • Apply to serial samples during and after treatment to quantify response

• Immunohistochemistry for tissue response assessment:

  • Standardize staining protocol across timepoints for consistent quantification

  • Develop digital image analysis algorithm for LRPAP1-reactive cell quantification

  • Create tissue microarrays from serial biopsies when available

  • Correlate with other response markers and clinical outcomes

• In vitro functional assays:

  • Measure changes in LRPAP1-induced BCR signaling before and after treatment

  • Assess cytotoxic effects of LRPAP1-based therapeutic constructs on patient-derived cells

  • Compare effectiveness of bispecific anti-CD3/LRPAP1