LRCH1 Antibody

What is LRCH1 and why is it relevant to immunological research?

LRCH1 is a member of the leucine-rich repeat (LRR) and calponin homology (CH) domain-containing protein family. It has been identified as a cytoskeletal regulator with significant roles in leukocyte function. The LRR domains facilitate protein-protein interactions while the CH domains mediate actin binding. LRCH1 has emerged as an important player in immune regulation, particularly in leukocyte migration and activation processes. Research has shown that LRCH1 functions as a negative regulator of natural killer (NK) cell function and suppresses migration of CD4+ T cells, making it relevant to studies of immune surveillance, host defense mechanisms, and inflammatory conditions such as ulcerative colitis .

Which epitopes of LRCH1 should researchers target when selecting antibodies?

When selecting antibodies against LRCH1, researchers should consider targeting conserved epitopes within the functional domains - specifically within the leucine-rich repeat (LRR) region for studying protein-protein interactions or within the calponin homology (CH) domain for investigating actin-binding functions. For immunoprecipitation studies, antibodies recognizing surface-exposed epitopes would be preferable. If studying LRCH1's role in CD4+ T cell migration, consider antibodies that can detect conformational changes in LRCH1 that may occur during interaction with PKCα, as this pathway has been implicated in LRCH1's inhibition of T cell migration toward CXCL12 .

How does LRCH1 expression vary across different immune cell populations?

LRCH1 expression varies significantly across immune cell populations and changes under inflammatory conditions. In monocyte-derived dendritic cells, LRCH1 expression is regulated through microRNA-155-mediated feedback loops following lipopolysaccharide (LPS) stimulation . LRCH1 is expressed in CD4+ T cells, with significantly decreased expression observed in CD4+ T cells from ulcerative colitis patients compared to healthy controls . In microglia, LRCH1 expression is downregulated following spinal cord injury . Natural killer cells also express LRCH1, where it functions as a negative regulator of cytotoxicity and cytokine production . This differential expression pattern suggests cell type-specific antibody validation is essential when studying LRCH1 in different immune contexts.

What are the optimal immunohistochemistry protocols for LRCH1 detection in tissue samples?

For optimal LRCH1 detection in tissue samples using immunohistochemistry, follow this validated protocol:

• Fix tissue samples and embed in paraffin following standard protocols

• Section tissues at 4-5 μm thickness

• Deparaffinize and rehydrate sections

• Perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 20 minutes

• Block endogenous peroxidase with Envision flex peroxidase-blocking reagent for 10 minutes

• Block non-specific binding with 5% normal serum

• Incubate with rabbit anti-human LRCH1 antibody (dilution 1:200) at 4°C overnight

• Wash thoroughly with PBS (3×5 minutes)

• Incubate with HRP-conjugated anti-rabbit secondary antibody at room temperature for 60 minutes

• Develop color with 3,3'-diaminobenzidine (DAB)

• Counterstain with hematoxylin

• Mount and visualize under light microscopy

This protocol has been successfully used to demonstrate decreased LRCH1 expression in colonic mucosa from patients with active ulcerative colitis .

How can researchers effectively use LRCH1 antibodies in flow cytometry applications?

For effective flow cytometry applications using LRCH1 antibodies:

• Harvest cells of interest (e.g., peripheral blood mononuclear cells, CD4+ T cells)

• Wash cells in PBS containing 2% FBS

• For surface staining: Incubate cells with fluorochrome-conjugated LRCH1 antibody (or primary antibody followed by labeled secondary antibody) at 4°C for 30 minutes in the dark

• For intracellular staining: Fix cells with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100 or commercial permeabilization buffer, then incubate with LRCH1 antibody

• Include appropriate isotype controls

• Analyze using multiparameter flow cytometry

When analyzing LRCH1 expression in CD4+ T cells, consider co-staining with markers of T cell activation (CD25, CD69) and differentiation (CD45RA, CD45RO) to correlate LRCH1 expression with functional states. Previous research has demonstrated LRCH1 expression differences in CD4+ T cells from ulcerative colitis patients compared to healthy controls, suggesting potential diagnostic applications .

What controls should be included when validating LRCH1 antibodies for Western blot?

When validating LRCH1 antibodies for Western blot, the following controls are essential:

• Positive control: Lysates from tissues or cell types known to express LRCH1 (e.g., PBMCs, NK cells, or colonic tissue from healthy individuals)

• Negative control:

  • LRCH1 knockout cell line (e.g., LRCH1 Knockout HeLa cell line)

  • Cells with LRCH1 knockdown using siRNA or shRNA

• Loading control: Probing for housekeeping proteins (β-actin, GAPDH) to ensure equal loading

• Peptide competition: Pre-incubation of antibody with immunizing peptide to confirm specificity

• Multiple antibodies: Using antibodies targeting different LRCH1 epitopes to confirm band identity

For quantitative comparisons, include a standard curve using recombinant LRCH1 protein at known concentrations. Previous studies have successfully used Western blot to demonstrate decreased LRCH1 protein expression in inflamed colonic mucosa from ulcerative colitis patients compared to healthy controls .

How can researchers effectively use LRCH1 knockout models to study antibody specificity and LRCH1 function?

To effectively use LRCH1 knockout models for antibody validation and functional studies:

• Antibody validation:

  • Compare antibody staining patterns between wild-type and LRCH1 knockout cells using Western blot, immunocytochemistry, and flow cytometry

  • Absence of signal in knockout cells confirms antibody specificity

  • Use commercially available LRCH1 Knockout HeLa cell line as a negative control

• Functional validation:

  • Generate LRCH1 knockout in relevant cell types (e.g., CD4+ T cells, NK cells) using CRISPR-Cas9

  • Perform rescue experiments by reintroducing wild-type LRCH1 or mutant variants

  • Compare phenotypes between knockout, rescue, and wild-type cells

• Experimental applications:

  • Investigate downstream signaling pathways affected by LRCH1 loss

  • Screen for compensatory mechanisms in LRCH1's absence

  • Study LRCH1's interaction partners using co-immunoprecipitation followed by mass spectrometry

Research using LRCH1-deficient NK92 cells has demonstrated increased cytotoxicity toward tumor cells and higher secretion of cytokines, revealing LRCH1's role as a negative regulator of NK cell function .

What are the optimal conditions for immunoprecipitation studies using LRCH1 antibodies?

For successful immunoprecipitation of LRCH1 and its binding partners:

• Cell lysis buffer optimization:

  • Use mild lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1% NP-40, pH 7.4) supplemented with protease and phosphatase inhibitors

  • For cytoskeletal interactions, include cytoskeleton stabilization buffer components

• Antibody selection and coupling:

  • Choose antibodies with high affinity for native LRCH1

  • Pre-couple antibodies to Protein A/G beads or use direct conjugation to minimize background

  • Consider epitope availability in the native protein conformation

• Immunoprecipitation protocol:

  • Pre-clear lysates with Protein A/G beads

  • Incubate cleared lysates with LRCH1 antibody-coupled beads overnight at 4°C with gentle rotation

  • Include appropriate controls (isotype control, input samples)

  • Wash extensively to reduce non-specific binding

• Analysis of interacting partners:

  • Elute bound proteins under native or denaturing conditions depending on downstream applications

  • Analyze by Western blot or mass spectrometry

This approach has successfully identified LRCH1's interaction with LAT (linker for activation of T cells) in CD8+ T cells, demonstrating how LRCH1 inhibits T cell receptor signaling by promoting LAT endocytosis and degradation .

How can researchers quantitatively compare LRCH1 expression across different disease states using antibody-based techniques?

For quantitative comparison of LRCH1 expression across disease states:

• Sample collection and standardization:

  • Collect matched tissue samples or cells from patients and controls

  • Process all samples using identical protocols

  • Include internal controls for normalization

• Quantitative techniques:

  • Western blot: Use digital imaging and densitometry with internal loading controls

  • Quantitative immunohistochemistry: Employ digital image analysis with standardized staining procedures

  • Flow cytometry: Measure mean fluorescence intensity with calibration beads

  • ELISA: Develop sandwich ELISA for LRCH1 quantification in serum or lysates

• Statistical analysis:

  • Use appropriate statistical tests for comparison between groups

  • Account for confounding variables through multivariate analysis

  • Correlate LRCH1 expression with disease severity indices

Studies have demonstrated that LRCH1 mRNA expression in colonic mucosa from ulcerative colitis patients negatively correlates with disease activity indices (Mayo index: r = -0.7014, p < 0.01; UCEIS: r = -0.6514, p < 0.001), suggesting LRCH1's potential as a biomarker .

How should researchers address inconsistent results when using different LRCH1 antibodies?

When faced with inconsistent results using different LRCH1 antibodies:

• Epitope comparison:

  • Compare the epitopes recognized by each antibody

  • Different domains may show variable accessibility in different experimental conditions

  • Consider post-translational modifications that might affect epitope recognition

• Validation strategies:

  • Test antibodies in LRCH1 knockout models to confirm specificity

  • Perform peptide competition assays

  • Compare antibody performance across multiple techniques (Western blot, IHC, flow cytometry)

• Experimental conditions:

  • Optimize fixation and antigen retrieval methods for each antibody

  • Test different blocking reagents to reduce non-specific binding

  • Adjust antibody concentration and incubation conditions

• Data interpretation:

  • Consider that differences might reflect biologically relevant LRCH1 isoforms or modifications

  • Use multiple antibodies targeting different epitopes for comprehensive analysis

  • Report all antibodies used and their validation data in publications

What are the common pitfalls in analyzing LRCH1 expression in immune cells and how can they be overcome?

Common pitfalls and solutions when analyzing LRCH1 expression in immune cells:

Studies have shown that LRCH1 expression is regulated under inflammatory conditions, with expression levels changing in response to stimuli like LPS or infection with various pathogens . This dynamic regulation necessitates careful experimental design and interpretation.

How can researchers distinguish between specific and non-specific binding when using LRCH1 antibodies in complex tissue samples?

To distinguish between specific and non-specific binding in complex tissue samples:

• Essential controls:

  • Isotype control antibodies to assess Fc receptor binding

  • Secondary antibody-only controls to detect non-specific secondary binding

  • Peptide competition to confirm epitope specificity

  • LRCH1 knockout or knockdown tissues as negative controls

• Optimization strategies:

  • Titrate antibody concentration to minimize background

  • Optimize blocking conditions using different blocking agents (BSA, normal serum, commercial blockers)

  • Include detergents in washing buffers to reduce hydrophobic interactions

  • Use antigen retrieval methods appropriate for the fixation method

• Advanced approaches:

  • Dual labeling with antibodies against known LRCH1-interacting proteins

  • Proximity ligation assay to detect specific protein-protein interactions

  • Multi-spectral imaging to distinguish autofluorescence from specific signal

  • Correlative microscopy combining immunolabeling with other techniques

Immunohistochemistry protocols for LRCH1 detection have been successfully implemented in studies of colonic tissue from ulcerative colitis patients, demonstrating specific staining of LRCH1-positive cells in the lamina propria .

How can antibody phage display technology be utilized to develop novel antibodies against specific LRCH1 epitopes?

Antibody phage display (APD) offers a powerful approach for developing novel LRCH1-specific antibodies:

• Library construction:

  • Start with high-quality RNA from an appropriate source (e.g., PBMCs from healthy donors)

  • Reverse transcribe to cDNA and PCR-amplify variable heavy (VH) and variable light (VL) chains

  • Use primer sets specific for different VH and VL gene families to capture the full immunoglobulin repertoire

  • Ligate PCR products into phage display vectors

• Selection strategy for LRCH1-specific antibodies:

  • Express recombinant LRCH1 protein or specific domains as selection targets

  • Perform cyclic panning against immobilized LRCH1 protein

  • Increase stringency in successive rounds to isolate high-affinity binders

  • Screen for domain-specific binding using truncated LRCH1 variants

• Antibody characterization:

  • Express selected scFv clones as soluble proteins

  • Evaluate binding affinity, specificity, and epitope recognition

  • Convert promising candidates to full IgG format if needed

The APD approach has been successfully used to isolate disease-specific antibodies in autoimmune conditions, demonstrating its potential for developing research and diagnostic tools .

What are the methodological considerations when using LRCH1 antibodies to investigate its role in CD4+ T cell migration?

When investigating LRCH1's role in CD4+ T cell migration using antibodies:

• Experimental design:

  • Isolate CD4+ T cells using magnetic separation or flow cytometry

  • Confirm LRCH1 expression levels by Western blot or flow cytometry

  • Use Transwell migration assays with CXCL12 as chemoattractant

  • Compare migration between wild-type cells, LRCH1-overexpressing cells, and LRCH1-knockdown cells

• Antibody applications:

  • Use blocking antibodies to inhibit LRCH1 function in migration assays

  • Employ antibodies for live cell imaging to track LRCH1 localization during migration

  • Apply proximity ligation assays to detect LRCH1 interactions with PKCα

  • Develop phospho-specific antibodies to monitor LRCH1 activation state

• Controls and validation:

  • Include isotype control antibodies in functional assays

  • Validate antibody specificity using LRCH1 knockout cells

  • Perform rescue experiments with LRCH1 constructs resistant to knockdown

Research has demonstrated that LRCH1 inhibits the migration of CD4+ T cells toward CXCL12 through a PKCα-dependent mechanism, highlighting the importance of this pathway in T cell trafficking regulation .

How can researchers reconcile conflicting data about LRCH1 function across different immune cell types?

To reconcile conflicting data about LRCH1 function across immune cell types:

• Systematic comparative analysis:

  • Design parallel experiments in multiple cell types using identical protocols

  • Compare LRCH1 expression levels, subcellular localization, and interaction partners

  • Analyze different LRCH1 isoforms and post-translational modifications

• Mechanistic investigations:

  • Map cell type-specific signaling pathways using phosphoproteomics

  • Identify cell type-specific LRCH1 binding partners through immunoprecipitation-mass spectrometry

  • Compare transcriptional responses to LRCH1 modulation across cell types

• Integrated approaches:

  • Develop conditional knockout models targeting specific immune cell populations

  • Employ single-cell analyses to detect heterogeneity within cell populations

  • Use systems biology approaches to model LRCH1 function in different cellular contexts

Research has revealed seemingly contradictory roles for LRCH1 across immune cells: it negatively regulates NK cell function through SFK-dependent pathways, inhibits microglial activation via MAPK and ERK1/2 pathways, and suppresses CD4+ T cell migration through PKCα . These diverse functions likely reflect cell type-specific interaction partners and signaling contexts.

How might LRCH1 antibodies be utilized to develop diagnostic tools for inflammatory bowel diseases?

LRCH1 antibodies show potential for developing diagnostic tools for inflammatory bowel diseases (IBD):

• Diagnostic applications:

  • Develop immunohistochemistry panels including LRCH1 for biopsy evaluation

  • Create flow cytometry assays to assess LRCH1 expression in peripheral blood CD4+ T cells

  • Design ELISA or multiplex assays to measure LRCH1 in patient serum or plasma

• Clinical correlation studies:

  • Evaluate LRCH1 expression correlation with disease activity indices (Mayo score, UCEIS)

  • Compare LRCH1 levels between UC, Crohn's disease, and non-IBD inflammatory conditions

  • Monitor LRCH1 expression during treatment response

• Validation requirements:

  • Perform large-scale studies across diverse patient populations

  • Standardize specimen collection and processing protocols

  • Establish reference ranges for different detection methods

Research has demonstrated that LRCH1 expression is significantly decreased in colonic mucosa and PBMCs from patients with active ulcerative colitis, and that expression levels negatively correlate with disease activity (r = -0.7014 with Mayo index, r = -0.6514 with UCEIS), suggesting potential diagnostic value .

What experimental approaches would best elucidate the structural changes in LRCH1 during immune cell activation and migration?

To elucidate structural changes in LRCH1 during immune cell activation and migration:

• Advanced microscopy techniques:

  • Super-resolution microscopy to visualize LRCH1 distribution during migration

  • FRET-based approaches to detect conformational changes upon activation

  • Live cell imaging with fluorescently tagged LRCH1 to track dynamic changes

• Biochemical approaches:

  • Limited proteolysis to identify exposed regions in different activation states

  • Hydrogen-deuterium exchange mass spectrometry to map structural dynamics

  • Crosslinking mass spectrometry to capture transient interactions

• Structure-function analysis:

  • Generate domain-specific antibodies to probe accessibility changes

  • Create LRCH1 mutants lacking specific domains or interaction motifs

  • Perform in silico molecular dynamics simulations based on experimental data

These approaches would help understand how LRCH1 regulates CD4+ T cell migration through PKCα-dependent mechanisms and how it interacts with proteins like LAT in T cells to modulate TCR signaling .