LRRC18 Antibody

What is LRRC18 and where is it predominantly expressed?

LRRC18 (Leucine Rich Repeat Containing 18) is a 261 amino acid protein (29.6 kDa) that contains 7 leucine-rich repeats and belongs to the LRR superfamily. While its precise function remains under investigation, current evidence suggests involvement in the regulation of spermatogenesis and sperm maturation. The protein localizes primarily in the cytoplasm and is predominantly expressed in male reproductive tissues, particularly the testis . This expression pattern makes LRRC18 a potentially important target for studying male reproductive biology and fertility. The protein has a calculated molecular weight of 30 kDa, which aligns with its observed molecular weight in experimental conditions .

What applications are LRRC18 antibodies suitable for?

LRRC18 antibodies have been validated for multiple research applications, with varying efficacy depending on the specific antibody clone and preparation. The most common applications include:

It's important to note that optimal dilutions and conditions may vary between different antibody preparations and should be determined empirically for each experimental system .

How should LRRC18 antibodies be stored to maintain reactivity?

Proper storage is critical for maintaining antibody functionality. For most LRRC18 antibodies, the recommended storage conditions are:

• Store at -20°C for long-term preservation

• For certain preparations, storage at -80°C may be recommended for extended periods

• Antibodies are typically stable for one year after shipment when stored properly

• Aliquoting is generally unnecessary for -20°C storage of preparations containing 50% glycerol

• Some preparations contain 0.1% BSA or other stabilizers in smaller volume formats

• Avoid repeated freeze-thaw cycles as this can compromise antibody performance

The standard storage buffer for many LRRC18 antibodies consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . This formulation helps maintain antibody stability during storage periods.

What is the optimal antigen retrieval method for LRRC18 immunohistochemistry?

When performing immunohistochemistry with LRRC18 antibodies on formalin-fixed, paraffin-embedded tissues, antigen retrieval is a critical step for exposing epitopes masked by fixation. Based on technical documentation, the recommended antigen retrieval methods are:

• Primary recommendation: TE buffer at pH 9.0

• Alternative method: Citrate buffer at pH 6.0

The selection between these methods may depend on tissue type, fixation duration, and the specific antibody being used. For human testis tissue specifically, which is a primary tissue type for LRRC18 expression studies, the TE buffer method at pH 9.0 has shown superior results in retrieving LRRC18 epitopes without compromising tissue morphology . Optimization of heating time and temperature is recommended for each specific tissue sample type.

How do different epitope targets affect LRRC18 antibody specificity?

Several LRRC18 antibodies target different regions of the protein, which can significantly impact their specificity and application suitability:

• C-terminal region targeting (AA 156-185): Antibodies targeting this region, such as ABIN953212, show reactivity with both human and mouse samples . These antibodies are particularly useful for Western blotting and paraffin-embedded immunohistochemistry applications.

• Full-length protein targeting (AA 1-261): Antibodies generated against the complete protein sequence typically demonstrate broader epitope recognition but may show more cross-reactivity .

• Mid-region targeting: Some antibodies target the middle portion of the protein (e.g., AA 60-109), which can offer different specificity profiles across species including human, mouse, dog, horse, rabbit, and monkey .

The choice of epitope target should be determined by the specific experimental question, the species under investigation, and whether native or denatured protein detection is required. For studying protein-protein interactions, antibodies targeting regions outside of interaction domains would be preferable.

What experimental controls should be implemented when using LRRC18 antibodies?

Rigorous experimental controls are essential for generating reliable results with LRRC18 antibodies:

• Positive tissue controls: Mouse or human testis tissue should be used as a positive control, as these have been validated for LRRC18 expression .

• Negative tissue controls: Tissues not known to express LRRC18 should be included to confirm antibody specificity.

• Blocking peptide controls: For antibodies raised against synthetic peptides, including a competition assay with the immunizing peptide can confirm binding specificity.

• Isotype controls: Using a non-specific IgG from the same host species (rabbit for most LRRC18 antibodies) at equivalent concentrations can help identify non-specific binding .

• Knockout/knockdown validation: Where available, tissue or cells with LRRC18 gene knockout or knockdown provide the most stringent specificity control.

• Dilution series: Testing multiple antibody dilutions helps establish the optimal signal-to-noise ratio for each experimental system .

For immunohistochemistry specifically, appropriate antigen retrieval controls should also be included to distinguish between true negative results and technical failures in epitope exposure.

How can LRRC18 antibodies be used to investigate spermatogenesis?

Given LRRC18's putative role in spermatogenesis and sperm maturation , antibodies against this protein represent valuable tools for reproductive biology research:

• Developmental expression profiling: Using immunohistochemistry with LRRC18 antibodies on testis tissue sections at different developmental stages can reveal temporal expression patterns during spermatogenesis.

• Cell type-specific localization: Double immunofluorescence staining with LRRC18 antibodies and markers for specific testicular cell populations (spermatogonia, spermatocytes, spermatids, Sertoli cells) can identify which cell types express the protein.

• Subcellular localization studies: Immunofluorescence microscopy with LRRC18 antibodies can determine the precise subcellular compartments where the protein functions, building on existing knowledge of its cytoplasmic localization .

• Protein-protein interaction studies: Immunoprecipitation with LRRC18 antibodies followed by mass spectrometry can identify binding partners in testicular tissue, providing insights into functional pathways .

• Expression changes in pathological conditions: Comparing LRRC18 expression levels and patterns between normal and pathological testis samples using Western blotting and immunohistochemistry may reveal associations with specific fertility disorders.

For these applications, careful antibody validation and optimization of protocols for testicular tissue are essential for generating reliable data.

What troubleshooting approaches are recommended for weak or non-specific LRRC18 signals?

When encountering suboptimal results with LRRC18 antibodies, several strategies can improve specific signal detection:

For Western Blotting:

• Increase protein loading (up to 30-50 μg of testis tissue lysate)

• Adjust antibody concentration within the recommended range (1:500-1:1000)

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

• Optimize blocking conditions to reduce background

• Use fresh tissue samples, as LRRC18 may be sensitive to degradation

For Immunohistochemistry:

• Compare both recommended antigen retrieval methods (TE buffer pH 9.0 and citrate buffer pH 6.0)

• Adjust antibody concentration across the recommended range (1:20-1:200)

• Increase incubation time with primary antibody

• Test different detection systems (HRP-polymer vs. biotin-streptavidin)

• Ensure tissues were promptly and properly fixed

For Immunoprecipitation:

• Increase antibody amount within the recommended range (0.5-4.0 μg)

• Optimize lysis buffer composition for testis tissue

• Extend incubation time with antibody-bead complex

• Perform pre-clearing of lysates to reduce non-specific binding

Each antibody preparation may require specific optimization, and it is recommended to titrate reagents in each testing system to obtain optimal results .

How can expression profiling with LRRC18 antibodies be integrated with genomic data?

Integrating protein expression data from LRRC18 antibody studies with genomic information can provide comprehensive insights into LRRC18 biology:

• Correlation with SNP variants: Expression levels of LRRC18 detected by antibodies can be correlated with specific SNP variants to identify potential genetic factors influencing protein expression, similar to approaches used in other integrative genomic studies .

• eQTL analysis: Quantitative immunohistochemistry or Western blot data can be integrated with expression quantitative trait loci (eQTL) data to identify genetic variants that influence LRRC18 expression levels.

• Multi-omics integration: Data from LRRC18 antibody-based assays can be combined with transcriptomic, proteomic, and epigenomic datasets to create comprehensive models of LRRC18 regulation.

• Phenotype correlation: LRRC18 expression levels determined by antibody-based methods can be correlated with reproductive phenotypes to establish potential functional relationships.

This integrative approach was successfully employed in genome-wide studies like the one described in the search results, where expression profile analysis was used to pinpoint genes influenced by specific antibody-associated SNPs . Similar methodologies could be applied to study LRRC18 in reproductive contexts.

What are the species cross-reactivity profiles of different LRRC18 antibodies?

The species cross-reactivity of LRRC18 antibodies varies depending on the epitope target and antibody preparation:

When selecting an LRRC18 antibody for cross-species studies, the epitope conservation between species should be evaluated. For comparative studies between human and mouse reproductive systems, antibodies targeting the C-terminal region (AA 156-185) have demonstrated consistent reactivity . For broader cross-species applications, antibodies targeting the mid-region (AA 60-109) may offer wider reactivity .

How do conjugated forms of LRRC18 antibodies compare in research applications?

Various conjugated forms of LRRC18 antibodies are available for specialized applications:

• Unconjugated antibodies: Most versatile, suitable for Western blot, immunoprecipitation, and immunohistochemistry with secondary detection systems .

• HRP-conjugated antibodies: Eliminate the need for secondary antibodies in Western blotting and ELISA, potentially reducing background and cross-reactivity issues . These are particularly useful for high-throughput screening applications.

• FITC-conjugated antibodies: Enable direct detection in flow cytometry and immunofluorescence microscopy without secondary antibodies .

• Biotin-conjugated antibodies: Offer signal amplification options through streptavidin-based detection systems, potentially increasing sensitivity in ELISA and immunohistochemistry applications .

Each conjugation type has specific advantages and limitations. For instance, while HRP-conjugated antibodies simplify workflows, they may have reduced shelf life compared to unconjugated versions. Similarly, fluorophore-conjugated antibodies eliminate secondary antibody steps but may have lower signal strength compared to amplified detection systems.

What sample preparation methods are optimal for LRRC18 detection in testis tissue?

Given LRRC18's predominant expression in testicular tissue, optimized sample preparation is crucial:

For Western Blotting:

• Fresh tissue extraction is preferred to minimize protein degradation

• Homogenization in RIPA buffer containing protease inhibitors

• Adequate sonication to disrupt cellular structures

• Centrifugation at high speed (≥12,000 × g) to clear debris

• Protein concentration determination before loading

For Immunohistochemistry:

• Prompt fixation in 10% neutral buffered formalin (12-24 hours)

• Careful processing to prevent over-fixation which could mask epitopes

• Paraffin embedding with controlled temperatures to prevent antigen degradation

• Sectioning at 4-5 μm thickness for optimal antibody penetration

• Storage of slides at 4°C if not used immediately

For Immunoprecipitation:

• Gentle lysis conditions to preserve protein-protein interactions

• Pre-clearing of lysates with protein A/G beads to reduce background

• Adequate protein concentration (1.0-3.0 mg of total protein)

• Optimal antibody amount (0.5-4.0 μg per immunoprecipitation)

For all applications, using testis tissue from appropriate developmental stages is important, as LRRC18 expression may vary during development and spermatogenesis.

How might LRRC18 antibodies contribute to understanding male infertility?

LRRC18 antibodies hold significant potential for advancing our understanding of male reproductive disorders:

• Biomarker development: Quantitative assessment of LRRC18 expression patterns in testicular biopsies from fertile versus infertile men could identify potential biomarkers for specific types of male infertility.

• Pathophysiological mechanisms: Systematic immunohistochemical studies using LRRC18 antibodies across different infertility disorders may reveal altered expression or localization patterns associated with specific pathologies.

• Functional studies: Using LRRC18 antibodies in combination with other molecular techniques could help elucidate the protein's role in spermatogenesis and sperm maturation, potentially revealing new therapeutic targets.

• Genetic variation impact: Integrating LRRC18 antibody-based expression studies with genetic analysis could identify how specific genetic variants affect LRRC18 expression and function in infertile populations.

• Therapy monitoring: In experimental therapeutic interventions for male infertility, LRRC18 antibodies could be used to monitor changes in protein expression as a measure of treatment efficacy.

The putative role of LRRC18 in spermatogenesis and sperm maturation makes it a particularly relevant target for investigating disorders of male reproductive function.

What are the recommended approaches for validating novel LRRC18 antibodies?

For researchers developing or characterizing new LRRC18 antibodies, a comprehensive validation approach should include:

• Western blot analysis: Confirming detection of a specific band at the expected molecular weight (30 kDa) in testis tissue .

• Peptide competition: Demonstrating signal elimination when the antibody is pre-incubated with the immunizing peptide or recombinant protein.

• Knockout/knockdown controls: Showing reduced or absent signal in tissues or cells with LRRC18 gene knockout or knockdown.

• Immunoprecipitation followed by mass spectrometry: Confirming that the antibody specifically pulls down LRRC18 protein.

• Cross-reactivity testing: Evaluating specificity across multiple species if cross-species reactivity is claimed.

• Application-specific validation: Testing the antibody in all intended applications (WB, IHC, IP, ELISA) with appropriate positive and negative controls.

• Epitope mapping: Determining the precise region of LRRC18 recognized by the antibody, which helps predict potential cross-reactivity.

• Reproducibility assessment: Ensuring consistent performance across different lots and in different laboratories.

This rigorous validation approach helps ensure that experimental results obtained with new LRRC18 antibodies are reliable and reproducible across different research contexts.