LRRC20 Antibody

What is LRRC20 protein and what biological roles does it play?

LRRC20 (Leucine-rich repeat-containing protein 20) is a cell surface molecule involved in various cellular processes, particularly in cellular regulation and signaling pathways. The protein contains leucine-rich repeat domains, which typically facilitate protein-protein interactions . With a calculated molecular weight of 21 kDa (though observed at 21-25 kDa range in various assays), LRRC20 has been detected in multiple human tissues including stomach, colon, and testis . While the complete functional characterization of LRRC20 remains an ongoing area of investigation, its structural features suggest potential roles in cellular communication and signal transduction that make it a valuable target for molecular biology research.

What types of LRRC20 antibodies are available for research applications?

Currently, researchers have access to several LRRC20 antibodies optimized for different experimental approaches. The primary types include polyclonal antibodies such as:

• Rabbit polyclonal antibodies (e.g., PACO38342, 25562-1-AP) that recognize human and mouse LRRC20

• Unconjugated antibodies validated for multiple applications including Western blotting, immunohistochemistry, and immunofluorescence

These antibodies are typically generated using recombinant human LRRC20 protein fragments or fusion proteins as immunogens, such as the recombinant human LRRC20 protein (1-184AA) used for PACO38342 antibody production . The polyclonal nature of current commercially available antibodies provides good sensitivity but researchers should be aware that specificity confirmation is essential for precise experimental outcomes.

What are the validated applications for LRRC20 antibodies?

The following table summarizes the validated applications and recommended dilutions for LRRC20 antibodies based on technical documentation:

These applications have been experimentally validated using specific cell lines and tissue samples. Western blotting has successfully detected LRRC20 in NIH/3T3 cells and Y79 cells, while IHC has been validated in human stomach cancer tissue, colon tissue, and testis tissue . For immunofluorescence, A431 cells have been confirmed as suitable samples for detection .

What are the optimal conditions for Western blotting with LRRC20 antibodies?

For optimal Western blot results with LRRC20 antibodies, researchers should consider the following protocol guidelines:

• Sample preparation: Standard cell lysis in RIPA or similar buffer with protease inhibitors

• Protein loading: 20-50 μg of total protein per lane is typically sufficient

• Gel percentage: 12-15% SDS-PAGE gels are recommended due to the relatively small size of LRRC20 (21-25 kDa)

• Transfer conditions: Standard wet or semi-dry transfer protocols are suitable

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation: Dilute LRRC20 antibody to 1:500-1:1000 for 25562-1-AP or 1:1000-1:5000 for PACO38342 in blocking buffer; incubate overnight at 4°C

• Secondary antibody: Anti-rabbit HRP-conjugated antibody at appropriate dilution

• Expected band size: 21-25 kDa

Positive controls such as NIH/3T3 or Y79 cell lysates should be included to confirm antibody performance . Multiple washing steps with TBST between each incubation step are essential to reduce background signal.

What protocols are recommended for LRRC20 antibody use in immunohistochemistry?

For immunohistochemical detection of LRRC20, the following methodology is recommended:

• Tissue preparation: Formalin-fixed, paraffin-embedded tissue sections (4-6 μm thickness)

• Antigen retrieval: Heat-mediated antigen retrieval is critical

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

• Blocking: 10% normal serum in PBS for 30 minutes

• Primary antibody dilution:

  • 25562-1-AP: 1:500-1:2000

  • PACO38342: 1:20-1:200

• Incubation time: Overnight at 4°C or 1-2 hours at room temperature

• Detection system: Standard HRP-conjugated secondary antibody with DAB substrate

• Counterstaining: Hematoxylin recommended for nuclear visualization

Validated positive control tissues include human stomach cancer tissue, human colon tissue, and human testis tissue . The immunohistochemistry data shows clear membrane and cytoplasmic staining patterns in positive tissues, providing good contrast for effective visualization.

How should LRRC20 antibodies be stored to maintain optimal reactivity?

Proper storage is essential for maintaining antibody integrity and performance. For LRRC20 antibodies:

• Storage temperature: Store at -20°C. Long-term stability has been validated for up to one year after shipment when stored properly

• Storage buffer:

  • 25562-1-AP: PBS with 0.02% sodium azide and 50% glycerol pH 7.3

  • PACO38342: 50% Glycerol, 0.01M PBS, PH 7.4 with 0.03% Proclin 300 as preservative

• Aliquoting: For 25562-1-AP, aliquoting is noted as unnecessary for -20°C storage

• Freeze-thaw cycles: Minimize repeated freeze-thaw cycles to prevent protein degradation

• Working solution: Once diluted for experimental use, store at 4°C and use within 24 hours

Proper storage conditions ensure consistent results across experiments and maximize the usable lifespan of the antibody reagent.

How can researchers validate the specificity of LRRC20 antibodies?

Antibody specificity validation is critical for meaningful research results. For LRRC20 antibodies, consider these validation approaches:

• Positive and negative controls: Include known positive cell lines (NIH/3T3, Y79, A431 cells) and tissues (human stomach, colon, and testis) in parallel with predicted negative samples

• Molecular weight verification: Confirm that the observed band appears at the expected molecular weight (21-25 kDa for LRRC20)

• Peptide competition assay: Pre-incubate the antibody with excess immunogenic peptide before application; this should abolish specific binding

• Knockdown/knockout validation: Use siRNA-mediated knockdown or CRISPR-Cas9 knockout of LRRC20 to demonstrate reduced or absent signal

• Multiple antibody comparison: Compare results using alternative LRRC20 antibodies targeting different epitopes

• Mass spectrometry validation: For definitive identification, immunoprecipitate the protein and verify by mass spectrometry

These validation steps help establish confidence in experimental results and distinguish specific from non-specific signals.

What are the optimal approaches for co-localization studies involving LRRC20?

When designing co-localization experiments to investigate LRRC20's interactions with other proteins:

• Immunofluorescence optimization: For IF/ICC applications, use LRRC20 antibody at 1:50-1:500 dilution with appropriate fluorophore-conjugated secondary antibodies

• Cell models: A431 cells have been validated for immunofluorescence detection of LRRC20 and provide a reliable experimental model

• Fixation methods: Standard 4% paraformaldehyde fixation is effective for LRRC20 detection

• Counterstaining: Include nuclear staining (DAPI) and additional markers for subcellular compartments

• Confocal microscopy settings: Use appropriate filter settings to minimize bleed-through when performing multi-color imaging

• Z-stack acquisition: Collect optical sections to confirm true co-localization in three dimensions

• Co-localization analysis: Apply quantitative co-localization analysis using software such as ImageJ with appropriate plugins

These approaches facilitate reliable detection of LRRC20's subcellular localization and its potential interactions with other cellular components.

How should researchers interpret variations in LRRC20 banding patterns in Western blot?

The observed molecular weight of LRRC20 typically ranges from 21-25 kDa, which may vary slightly from the calculated 21 kDa . When interpreting Western blot results:

• Multiple bands: Consider potential post-translational modifications, alternative splicing, or proteolytic processing. LRRC20 may undergo phosphorylation or other modifications that alter migration

• Higher molecular weight bands: May indicate protein complexes that weren't fully denatured, dimerization, or potential cross-reactivity

• Lower molecular weight bands: Could represent degradation products or alternative isoforms

• Tissue-specific variations: Compare banding patterns across different tissue or cell types, as expression patterns may legitimately vary

• Reducing vs. non-reducing conditions: Consider running samples under both conditions to identify any disulfide-bond dependent structures

Always include positive controls (NIH/3T3 or Y79 cell lysates) alongside experimental samples to establish a reference banding pattern .

What cell and tissue types are optimal for studying LRRC20 expression?

Based on validation data, these biological samples are recommended for LRRC20 research:

When designing experiments, these validated models provide reliable baseline expression for comparative studies. The differential expression across tissues suggests potential tissue-specific functions of LRRC20 that may be worth investigating further in specialized research contexts.

What are common troubleshooting strategies for failed LRRC20 detection?

When experiments fail to detect LRRC20, consider these troubleshooting approaches:

• Western blot issues:

  • Increase protein loading (50-100 μg/lane)

  • Optimize primary antibody concentration

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

  • Enhance sensitivity with alternative detection systems

  • Ensure sample preparation preserves protein integrity

• IHC/IF problems:

  • Optimize antigen retrieval (test both recommended methods: TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Adjust antibody concentration (try a range of dilutions)

  • Extend primary antibody incubation time

  • Use amplification systems for low-abundance targets

  • Test different fixation protocols

• General considerations:

  • Verify target expression in your specific sample using reference databases

  • Check antibody viability (age, storage conditions)

  • Include established positive control samples

  • Test alternative antibody lots or sources

How can LRRC20 antibodies be utilized in high-throughput screening approaches?

For adapting LRRC20 detection to high-throughput applications:

• ELISA development: Both commercially available LRRC20 antibodies have been validated for ELISA applications, with PACO38342 recommended at 1:2000-1:10000 dilution

• Tissue microarray analysis: IHC protocols can be adapted for tissue microarrays to assess LRRC20 expression across multiple samples simultaneously

• Cell-based screening: Immunofluorescence protocols can be modified for automated microscopy platforms using validated cell models like A431

• Protein array applications: LRRC20 antibodies may be suitable for protein array applications to assess interaction partners

• Flow cytometry adaptation: Though not specifically validated in the provided documentation, polyclonal LRRC20 antibodies could potentially be adapted for flow cytometry following standard optimization protocols

These high-throughput approaches allow for more comprehensive analysis of LRRC20's role in different biological contexts and potential disease associations.

What considerations are important when designing antibody-based studies to investigate LRRC20's role in signaling pathways?

When investigating LRRC20's functional role in cellular signaling:

• Stimulation conditions: Design experiments that include relevant cellular stimuli to activate pathways potentially involving LRRC20

• Temporal analysis: Include multiple time points to capture dynamic changes in LRRC20 expression, localization, or modification

• Phosphorylation studies: Consider complementing total LRRC20 detection with phospho-specific antibodies if available, or use phosphoprotein enrichment followed by LRRC20 detection

• Co-immunoprecipitation: Use validated LRRC20 antibodies for IP to identify interaction partners under different cellular conditions

• Pathway perturbation: Combine LRRC20 detection with specific pathway inhibitors to map its relationship to established signaling cascades

• Quantitative analysis: Implement quantitative approaches (densitometry for Western blots, fluorescence intensity for IF) to measure changes in LRRC20 levels

These experimental design considerations help position LRRC20 within the broader context of cellular signaling networks and reveal its functional significance.