LRRC32 Antibody

What is LRRC32/GARP and why is it important in immunological research?

LRRC32/GARP is a transmembrane glycoprotein of 662 amino acids with its extracellular portion containing 20 leucine-rich repeats . It functions as a cell surface receptor primarily on regulatory T-lymphocytes (Treg cells), platelets, hepatic stellate cells, and certain cancer cells . LRRC32 has been demonstrated as a Treg-specific activation marker and plays a critical role in the surface expression of latent TGF-β by binding to the complex . This protein is particularly significant in immunological research because it helps identify potent regulatory T cell subsets - LRRC32+ Tregs demonstrate greater suppressive capacity than LRRC32- Tregs . Understanding LRRC32 expression and processing provides insight into Treg function and potential immunotherapeutic strategies targeting these cells . The protein's selective expression on activated FOXP3+ regulatory T cells makes it a valuable marker for distinguishing functionally suppressive Tregs from other T cell populations .

Sample preparation depends on whether you are detecting surface or intracellular LRRC32:

For surface expression detection in flow cytometry:

• Harvest cells and wash in PBS containing 1% BSA

• Block non-specific binding with appropriate isotype controls

• Stain with anti-LRRC32 antibody without permeabilization

• Wash and analyze by flow cytometry

For intracellular detection:

• First stain surface antigens

• Fix and permeabilize cells using a fixation permeabilization kit

• Block with isotype control antibody (e.g., 2.5 μg/ml of IgG2b) for 30 minutes to reduce non-specific binding

• Incubate with labeled anti-LRRC32 antibody

• Wash and analyze

For Western blot detection:

• Lyse cells in appropriate buffer (e.g., RIPA buffer)

• Reduce samples before loading (LRRC32 appears at approximately 80-85 kDa on gels)

• Use Immunoblot Buffer Group 1 for optimal results

• Transfer to PVDF membrane for best detection

When working with tissue sections for IHC, heat-induced epitope retrieval is crucial for optimal antibody binding, as demonstrated in mouse spleen tissue studies .

How do I differentiate between intracellular and surface expression of LRRC32?

Differentiating between intracellular and surface expression of LRRC32 requires specific experimental approaches:

For flow cytometry:

• Surface detection: Stain unfixed, non-permeabilized cells with anti-LRRC32 antibody

• Intracellular detection: After surface marker staining, fix and permeabilize cells before LRRC32 staining

• Compare the same cell population with both staining approaches to determine localization patterns

Studies have demonstrated that low levels of LRRC32 are present intracellularly in freshly isolated Tregs prior to activation, while surface expression occurs following activation . To accurately distinguish these populations, include appropriate controls and carefully analyze co-expression with other markers like CD25 and FoxP3 .

Microscopy techniques can also help determine localization:

• Immunofluorescence microscopy shows cytoplasmic localization in certain cell types like the bEnd.3 mouse endothelioma cell line

• Surface expression can be confirmed using non-permeabilized cells with confocal microscopy

• Comparing signal intensity between permeabilized and non-permeabilized samples helps quantify the relative distribution

Importantly, signal peptide cleavage is essential for surface expression of LRRC32, so mutations or inhibition of this process would prevent surface localization while potentially preserving intracellular expression .

What are the optimal conditions for detecting LRRC32 in Western blot applications?

For optimal Western blot detection of LRRC32, follow these guidelines:

When detecting LRRC32 in transfected samples, note that the protein may appear at a different molecular weight (~100 kDa) in Fc fusion constructs compared to the native protein (~72 kDa) . This size discrepancy should be considered when interpreting results from recombinant proteins versus endogenous expression .

For optimal sensitivity, protein concentration should be determined and standardized across lanes, and exposure times should be optimized to avoid signal saturation while maintaining clear band visualization .

How should I select the appropriate LRRC32 antibody for my specific research needs?

Selecting the appropriate LRRC32 antibody depends on your specific research application, target species, and experimental goals:

• Consider target species reactivity:

  • Some antibodies show cross-reactivity between human and mouse LRRC32

  • Certain antibodies may have partial cross-reactivity (~50%) with other species

  • Verify published validation data for your species of interest

• Match antibody type to application:

  • Polyclonal antibodies (e.g., 26021-1-AP) offer higher sensitivity but potentially lower specificity

  • Monoclonal antibodies (e.g., Plato-1) provide consistent results with high specificity

  • Consider using different antibody clones for validation experiments

• Evaluate epitope binding region:

  • Antibodies targeting different domains may detect different forms of the protein

  • Previous studies using antibodies against amino acids 296-308 failed to detect LRRC32 on some cell types

  • Consider antibodies recognizing the extracellular domain (e.g., Ile18-Asn628) for detecting native conformation

• Review validated applications in literature:

  • Some antibodies have been cited in published research for specific applications

  • Consider antibodies with demonstrated performance in your experimental system

  • Check if the antibody detects both glycosylated and non-glycosylated forms if relevant

• Match host species to your experimental system:

  • Rabbit, sheep, and mouse host antibodies are available

  • Choose a host that minimizes cross-reactivity with other reagents in your system

Always perform proper validation experiments including positive and negative controls to ensure the antibody performs as expected in your specific experimental conditions.

How can I use LRRC32 antibodies to investigate the relationship between LRRC32 and TGF-β signaling?

LRRC32 (GARP) plays a critical role in tethering latent TGF-β on the surface of activated regulatory T cells, making it an excellent target for investigating TGF-β pathways . Here are methodological approaches using LRRC32 antibodies:

• Co-localization studies:

  • Use dual immunofluorescence with anti-LRRC32 and anti-LAP (latency-associated peptide) antibodies

  • Analyze co-localization patterns using confocal microscopy

  • Quantify Pearson's correlation coefficients between signals

• Functional blockade experiments:

  • Use anti-LRRC32 antibodies to block the interaction between LRRC32 and latent TGF-β

  • Compare TGF-β signaling activities (e.g., Smad phosphorylation) in blocked versus unblocked conditions

  • Assess downstream functional effects on target cell populations

• Pull-down and co-immunoprecipitation:

  • Use LRRC32 antibodies for immunoprecipitation (1:200 dilution recommended)

  • Western blot for co-precipitated TGF-β complex components

  • Verify interactions using reciprocal co-immunoprecipitation

• Flow cytometry analysis:

  • Measure surface expression of LRRC32 and LAP-TGF-β using specific antibodies

  • Correlate expression levels with Treg suppressive function

  • Sort LRRC32+ and LRRC32- populations to compare TGF-β production and activity

This approach provides mechanistic insights into how LRRC32 contributes to Treg function through TGF-β presentation, as research has demonstrated that LRRC32 binds directly to TGF-β LAP and tethers latent TGF-β on activated Treg cell surfaces, which contributes to their suppressive capacity .

What strategies can be employed to study the role of LRRC32 in regulatory T cell function?

To comprehensively study LRRC32's role in regulatory T cell function, researchers can employ these methodological strategies:

• Comparative functional analysis of LRRC32+ and LRRC32- Treg populations:

  • Isolate fresh CD4+CD25hi T cells from peripheral blood

  • Sort into LRRC32+ and LRRC32- subpopulations using flow cytometry

  • Compare suppressive capacity using mixed lymphocyte reaction assays

  • Research has demonstrated that LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs

• Phenotypic characterization:

  • Analyze co-expression of LRRC32 with other Treg markers (FoxP3, CD25, CD62L)

  • Studies show LRRC32+ Tregs are distinct from LRRC32- Tregs in CD62L expression

  • Use multiparameter flow cytometry to create comprehensive phenotypic profiles

• Activation-dependent expression studies:

  • Monitor LRRC32 expression before and after Treg activation

  • Correlate expression levels with suppressive function

  • Investigate intracellular versus surface localization during activation stages

• Signal peptide processing analysis:

  • Use constructs with deleted putative signal peptide regions

  • Compare surface expression of wild-type versus mutant LRRC32

  • Research confirms that signal peptide cleavage is essential for surface expression

• Knockdown/inhibition experiments:

  • Use siRNA or lentiviral approaches targeting LRRC32

  • Evaluate impact on Treg suppressive capacity

  • Studies show inhibition of LRRC32 expression results in decreased suppressive capacity

These approaches should be combined with appropriate controls, including isotype antibody controls for flow cytometry and comparison with effector T cell populations that do not express LRRC32 .

How can I troubleshoot inconsistent or weak LRRC32 detection in my experiments?

When encountering inconsistent or weak LRRC32 detection, consider these methodological troubleshooting approaches:

• For Western blot inconsistencies:

  • Verify protein loading: LRRC32 has a calculated molecular weight of 72 kDa but is observed at ~80-85 kDa due to glycosylation

  • Ensure reducing conditions: Use proper reducing agents in sample buffer

  • Optimize antibody concentration: Test dilution ranges from 1:200-1:1000

  • Check positive controls: Use HUVEC cells or mouse spleen non-B cells as validated sources

  • Consider membrane type: PVDF membranes may provide better results than nitrocellulose

• For immunohistochemistry weak signals:

  • Optimize antigen retrieval: Try both TE buffer pH 9.0 and citrate buffer pH 6.0

  • Increase antibody concentration: Test higher concentrations within the 1:50-1:500 range

  • Extend incubation time: Consider overnight incubation at 4°C

  • Use amplification systems: Consider tyramide signal amplification for weak signals

  • Verify positive tissue controls: Human tonsillitis tissue and mouse spleen have confirmed expression

• For flow cytometry challenges:

  • Block non-specific binding: Pre-incubate with isotype control (2.5 μg/ml IgG2b)

  • Consider activation status: LRRC32 expression increases after Treg activation

  • Check for intracellular expression: Some cells may have primarily intracellular rather than surface expression

  • Optimize permeabilization: Different fixation/permeabilization protocols may affect epitope accessibility

  • Use fresh samples: LRRC32 expression may decrease with extended cell culture

• General considerations:

  • Antibody storage: Ensure proper storage at -20°C for long-term stability

  • Sample handling: Minimize freeze-thaw cycles for both antibodies and protein samples

  • Consider epitope accessibility: Previous studies using antibodies against amino acids 296-308 failed to detect LRRC32 in certain contexts

  • Test multiple antibody clones: Different antibodies may recognize different epitopes with varying accessibility

Always titrate antibodies in each testing system for optimal results, as recommended by manufacturers .

What are the best approaches for studying LRRC32 in non-immune cell types?

While LRRC32 is primarily studied in Tregs, investigating its role in non-immune cells requires specific methodological considerations:

• For endothelial cells and megakaryocytes:

  • Verify baseline expression using Western blot on HUVEC cells, a validated positive control

  • Use immunofluorescence for localization studies, as demonstrated in bEnd.3 mouse endothelioma cells

  • Apply 10 μg/mL antibody concentration for 3 hours at room temperature for optimal staining

  • Counterstain with DAPI to visualize nuclei and confirm cytoplasmic localization

• For platelet studies:

  • Consider that LRRC32 is expressed on adult platelets

  • Use flow cytometry with careful gating strategies for these small cellular fragments

  • Compare expression patterns with activation markers to determine correlation with platelet activation status

  • Implement appropriate platelet isolation protocols to minimize activation during preparation

• For cancer cells:

  • Screen cell lines for expression using Western blot before detailed studies

  • Compare expression levels with paired normal tissues when available

  • Correlate with TGF-β signaling activity using reporter assays

  • Consider analyzing LRRC32 expression in relation to tumor immunosuppressive mechanisms

• For hepatic stellate cells:

  • Use co-staining with stellate cell markers for verification

  • Analyze expression changes during stellate cell activation and fibrosis progression

  • Consider both surface and intracellular localization patterns

  • Correlate with TGF-β production and fibrogenic activity

In all non-immune cell studies, appropriate controls are essential:

• Include positive controls (Tregs or HUVEC cells) alongside test samples

• Use multiple detection methods to confirm expression (Western blot, flow cytometry, immunostaining)

• Verify specificity using competing peptides or knockout/knockdown approaches when possible

• Consider context-specific expression regulation that may differ from immune cells

How do I optimize antibody dilutions for different applications and sample types?

Optimal antibody dilution varies by application, sample type, and specific antibody clone. Here's a methodological approach to optimization:

For systematic optimization:

• Western blot:

  • Run parallel blots with the same samples at different antibody dilutions

  • Assess signal strength, background, and specificity at each dilution

  • Select dilution with highest signal-to-noise ratio and specific bands at expected molecular weight (80-85 kDa)

• Immunohistochemistry/Immunofluorescence:

  • Test a dilution series on positive control tissues (human tonsillitis, mouse spleen)

  • Evaluate specific staining versus background at each concentration

  • Consider both primary antibody incubation time and temperature

  • For immunofluorescence, 10 μg/mL for 3 hours at room temperature has been validated

• Flow cytometry:

  • Create titration curves using known positive cells

  • Calculate staining index (median positive/median negative) at each concentration

  • Select concentration with highest staining index

  • Consider blocking with isotype controls (2.5 μg/ml IgG2b) for 30 minutes to reduce background

Remember that optimal dilutions may need readjustment when:

• Switching between fresh and fixed tissues

• Moving between different detection systems

• Working with samples from different species

• Using new antibody lots

As noted in the product information, "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" .

What are the critical factors affecting the detection of LRRC32 in tissue sections?

Several critical factors significantly impact LRRC32 detection in tissue sections:

• Antigen retrieval method:

  • Heat-induced epitope retrieval is essential for optimal antibody binding

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • VisUCyte Antigen Retrieval Reagent-Basic has been validated for mouse spleen sections

• Fixation parameters:

  • Overfixation can mask epitopes and reduce antibody binding

  • Immersion fixation with paraffin embedding has been successfully used

  • Duration of fixation should be optimized for tissue type and thickness

• Antibody selection and concentration:

  • Different antibodies may detect different epitopes with varying accessibility

  • Concentration range for IHC: 1:50-1:500 or 0.3 μg/mL

  • Incubation time affects sensitivity (overnight at 4°C recommended for some protocols)

• Detection system selection:

  • HRP polymer detection systems provide enhanced sensitivity

  • Secondary antibody must match primary antibody host species

  • Chromogenic substrates (e.g., DAB) produce brown staining that contrasts well with hematoxylin counterstain

• Tissue-specific considerations:

  • LRRC32 expression varies by tissue type and activation state

  • Positive controls should include:

    • Human tonsillitis tissue

    • Mouse spleen tissue

  • Specific staining patterns vary:

    • Extracellular space localization observed in mouse spleen

    • Cytoplasmic localization in certain cell types

• Signal specificity verification:

  • Include negative controls (isotype antibody or secondary-only staining)

  • Use tissues known to lack LRRC32 expression as negative controls

  • Consider blocking peptide controls to confirm specificity

These factors should be systematically optimized when establishing LRRC32 detection protocols for tissue sections, with special attention to antigen retrieval methods which appear particularly critical for successful staining .

How can LRRC32 antibodies be used to identify and isolate therapeutically relevant regulatory T cell populations?

LRRC32 antibodies offer powerful tools for identifying and isolating therapeutically relevant Treg populations through these methodological approaches:

• Flow cytometry-based identification and sorting:

  • Use multi-parameter flow cytometry combining CD4, CD25, FoxP3, and LRRC32 markers

  • Implement the following gating strategy:

    • First gate on CD4+ lymphocytes

    • Then identify CD25hi population (top 5% of CD25+ cells)

    • Further characterize based on LRRC32 expression

  • Sort LRRC32+ and LRRC32- Treg subpopulations for functional studies or therapeutic applications

  • Research confirms LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs

• Functional verification of isolated populations:

  • Assess suppressive capacity in mixed lymphocyte reaction assays

  • Measure TGF-β production and presentation capacity

  • Evaluate stability of FoxP3 expression and suppressive phenotype

  • Compare gene expression profiles between LRRC32+ and LRRC32- Treg subsets

• Therapeutic applications:

  • Enrich for LRRC32+ Tregs in adoptive transfer protocols to enhance efficacy

  • Use LRRC32 as a quality control marker for manufactured Treg products

  • Monitor LRRC32 expression during ex vivo expansion to track functional potential

  • Correlate clinical outcomes with LRRC32 expression levels in administered Tregs

• Advanced isolation strategies:

  • Combine magnetic pre-enrichment of CD4+CD25+ cells with LRRC32-based FACS sorting

  • Develop GMP-compliant isolation protocols using clinical-grade antibodies

  • Implement closed-system isolation platforms for therapeutic applications

  • Consider activation-induced expression for expanding LRRC32+ populations

This approach helps "select for more potent Treg populations" as suggested by research demonstrating that "LRRC32 surface expression may be useful as a marker that selects for more potent Treg populations" .

What are the considerations for using LRRC32 antibodies in developing cancer immunotherapies?

LRRC32/GARP antibodies present unique opportunities and challenges for cancer immunotherapy development:

• Target validation strategies:

  • Assess LRRC32 expression in tumor-infiltrating Tregs versus peripheral Tregs

  • Analyze correlation between LRRC32+ Treg infiltration and patient outcomes

  • Determine LRRC32 expression on tumor cells themselves, as some cancer cells express LRRC32

  • Evaluate relationship between LRRC32 expression and TGF-β signaling in the tumor microenvironment

• Therapeutic antibody development considerations:

  • Select antibodies binding specific epitopes that block TGF-β presentation

  • Evaluate different antibody formats (IgG1, IgG4, F(ab')2) for optimal efficacy

  • Consider Fc engineering to enhance or suppress effector functions depending on mechanism of action

  • Test for cross-reactivity between human and mouse LRRC32 (~80% amino acid sequence identity) for translational studies

• Combination therapy approaches:

  • Test LRRC32-targeting agents with immune checkpoint inhibitors

  • Combine with TGF-β pathway inhibitors for potential synergistic effects

  • Evaluate sequential versus concurrent administration strategies

  • Monitor changes in tumor-infiltrating lymphocyte populations following treatment

• Monitoring and biomarker development:

  • Use flow cytometry to track LRRC32+ Tregs during treatment

  • Develop immunohistochemistry protocols for FFPE tumor samples

  • Correlate LRRC32 expression with other immunosuppressive markers

  • Monitor TGF-β activity as a pharmacodynamic endpoint

• Preclinical model selection:

  • Consider species cross-reactivity (~80% sequence identity between human and mouse LRRC32)

  • Validate antibody binding to mouse LRRC32 before selecting murine models

  • Consider humanized mouse models where appropriate

  • Verify LRRC32 expression patterns in model systems match human disease

These methodological considerations address the complex role of LRRC32 in regulating TGF-β bioavailability in the tumor microenvironment, which may contribute to immunosuppression and tumor progression through Treg-mediated suppression mechanisms .