LYRM4 Antibody, HRP conjugated

What is LYRM4 and why is it significant in hepatocellular carcinoma research?

LYRM4 is an essential iron-sulphur cluster biogenesis factor that maintains the stability and activity of the human cysteine desulfurase complex NFS1-LYRM4-ACP. Recent bioinformatics analyses and clinical specimen evaluations have demonstrated that LYRM4 is significantly overexpressed in liver hepatocellular carcinoma (LIHC) tissues compared to normal liver tissues . The protein plays a crucial role in iron-sulphur cluster (ISC) biosynthesis, which appears to be elevated in LIHC, potentially leading to ISC-dependent metabolic reprogramming. This metabolic shift may contribute to tumor progression, making LYRM4 a promising target for both diagnostic and therapeutic interventions .

The significance of LYRM4 in hepatocellular carcinoma is further underscored by its correlation with various clinical parameters. Research indicates that LYRM4 mRNA expression is related to clinical stratifications, prognosis, and survival outcomes in LIHC patients. Additionally, LYRM4 expression shows significant associations with ALT levels, tumor thrombus, and encapsulation in HBV-related LIHC patients, suggesting its potential utility as a biomarker .

How does HRP conjugation enhance LYRM4 antibody detection sensitivity?

Horseradish peroxidase (HRP) conjugation significantly enhances detection sensitivity through its enzymatic amplification properties. When HRP is conjugated to a LYRM4 antibody, it catalyzes the oxidation of substrates like diaminobenzidine (DAB) in the presence of hydrogen peroxide, producing a visible, stable precipitate at the site of antibody binding . This enzymatic reaction creates signal amplification that allows visualization of even low-abundance LYRM4 protein in tissue samples.

The LYNX Rapid Conjugation kit system enables directional covalent bonding of HRP to the antibody structure, which maintains antibody specificity while providing the enhanced detection capabilities . This directional conjugation preserves the antigen-binding capacity of the antibody, ensuring that detection sensitivity gains do not come at the cost of specificity. Researchers have utilized this enhanced sensitivity to detect subtle differences in LYRM4 expression between LIHC tissues and paired adjacent normal tissues, revealing correlations with clinical parameters that might otherwise be undetectable .

What are the fundamental principles of immunohistochemistry using LYRM4 antibodies?

Immunohistochemistry (IHC) for LYRM4 detection follows several critical steps to ensure specific and sensitive protein visualization. The process begins with proper tissue preparation, typically involving formalin-fixation and paraffin-embedding of liver tissue samples. For systematic analysis, tissue microarrays (TMAs) can be constructed from these samples .

The primary principles include:

• Antigen retrieval: This overcomes fixative-induced protein cross-linking that may mask LYRM4 epitopes.

• Blocking steps: This minimizes non-specific binding of the LYRM4 antibody.

• Primary antibody incubation: Tissues are incubated with LYRM4-specific antibodies (typically 1:100 dilution) at 4°C overnight .

• Secondary antibody application: Biotin-labeled goat anti-rabbit IgG is applied to bind the primary antibody.

• Signal development: Following incubation with streptavidin-biotin-peroxidase complex (SABC), the reaction is visualized using DAB substrate, which produces a brown precipitate at sites of LYRM4 expression .

• Counterstaining: Hematoxylin is used to visualize cell nuclei, providing context for LYRM4 localization.

Controls are essential for result validation, with PBS substituting for primary antibody serving as a negative control .

What experimental applications benefit most from using HRP-conjugated LYRM4 antibodies?

HRP-conjugated LYRM4 antibodies provide significant advantages in several research applications focused on hepatocellular carcinoma investigation:

• Tissue microarray analysis: HRP conjugation enables high-throughput screening of LYRM4 expression across multiple patient samples with consistent signal development, facilitating correlations with clinical parameters including age, serum low-density lipoprotein (LDL), and triglyceride (TG) content .

• Differential expression studies: The enhanced sensitivity allows precise discrimination between LYRM4 expression in LIHC tissues versus normal adjacent tissues, revealing that LYRM4 is significantly overexpressed in tumor tissues .

• Prognostic biomarker validation: The stable and quantifiable signal provided by HRP conjugation supports statistical analysis correlating LYRM4 expression with patient survival and other clinical outcomes .

• Immune infiltration assessment: HRP-conjugated antibodies can be used in multiplex IHC approaches to simultaneously evaluate LYRM4 expression and immune cell infiltration, helping to elucidate the relationship between LYRM4 and the tumor immune microenvironment .

• Mechanistic studies: When investigating the functional relationship between LYRM4 and iron-sulphur dependent proteins like POLD1 and PRIM2, HRP-conjugated antibodies provide reliable detection across diverse experimental conditions .

How can researchers correlate LYRM4 expression patterns with immune cell infiltration in hepatocellular carcinoma?

Researchers can employ a multi-dimensional approach to correlate LYRM4 expression with immune infiltration in hepatocellular carcinoma. The TIMER2.0 database provides a systematic framework for investigating these associations through Spearman correlation tests adjusted for tumor purity . Studies have revealed that LYRM4 expression exhibits significant positive correlations with six distinct immune cell types in the LIHC microenvironment, suggesting its potential role in modulating anti-tumor immunity.

To establish these correlations experimentally, researchers should:

• Perform multiplexed immunohistochemistry or immunofluorescence to simultaneously detect LYRM4 and immune cell markers in the same tissue sections.

• Conduct single-cell RNA sequencing to capture both LYRM4 expression and immune cell transcriptional signatures at cellular resolution.

• Analyze spatial relationships between LYRM4-expressing tumor cells and infiltrating immune cells using digital pathology platforms.

• Validate computational findings from databases like TIMER2.0 with matched patient cohorts, examining both LYRM4 expression and immune cell quantification .

• Utilize Kaplan-Meier survival analysis to evaluate how the combination of LYRM4 expression and immune infiltration levels impacts patient outcomes, as both factors have been strongly associated with prognosis .

What are the optimal buffer conditions for maintaining LYRM4 antibody activity during HRP conjugation?

The maintenance of LYRM4 antibody activity during HRP conjugation requires careful attention to buffer composition and pH. Optimal buffer conditions include:

• Buffer type: 10-50mM amine-free buffers such as HEPES, MES, MOPS, or phosphate are recommended. These buffers provide a stable environment for the conjugation reaction without interfering with the chemistry of the LYNX conjugation system .

• pH range: Maintaining pH between 6.5-8.5 is critical for preserving antibody structure while enabling efficient conjugation reactions .

• Components to avoid: Buffers containing nucleophilic components such as primary amines and thiols (including preservatives like thiomersal/thimerosal) should be strictly avoided as they may react with LYNX chemicals and compromise conjugation efficiency .

• Tris buffer considerations: While not ideal, moderate concentrations of Tris buffer (<20mM) may be tolerated if necessary .

• Sodium azide exclusion: As sodium azide is an irreversible inhibitor of HRP, it must be completely eliminated from antibody preparations prior to conjugation .

• Salt and sugar impact: EDTA and common non-buffering salts and sugars have minimal effect on conjugation efficiency and may be present in the buffer .

For optimal results, the LYRM4 antibody should be prepared at a concentration range of 0.5-5.0 mg/ml in the appropriate buffer before initiating the conjugation procedure .

How does LYRM4 overexpression influence iron-sulphur protein activity in hepatocellular carcinoma?

LYRM4 overexpression profoundly impacts iron-sulphur protein activity in hepatocellular carcinoma, driving metabolic adaptations that support tumor progression. Experimental evidence demonstrates that mitochondrial complex I and aconitate hydratase (ACO2) activities are significantly increased in LIHC cell lines compared to normal hepatocytes, indicating enhanced iron-sulphur cluster (ISC) biosynthesis .

This metabolic reprogramming occurs through several mechanisms:

• Enhanced ISC assembly: LYRM4 overexpression increases the efficiency of the NFS1-LYRM4-ACP complex, accelerating ISC formation and insertion into recipient proteins .

• Upregulation of key ISC-dependent proteins: The expression of iron-sulphur proteins POLD1 and PRIM2 is significantly elevated in LIHC tissue and correlates with poor prognosis, suggesting functional consequences of LYRM4 overexpression .

• Metabolic pathway alterations: Biological interaction and gene regulation network analyses reveal that LYRM4-associated genes predominantly participate in the citric acid cycle and oxidative phosphorylation, consistent with increased ACO2 activity .

• Regional metabolic adaptations: Changes in ISC-dependent protein activity are not limited to tumor tissue but may also occur in paracancerous tissues, indicating a field effect that could influence tumor microenvironment .

These findings suggest that targeting LYRM4 could potentially disrupt the iron-sulphur protein-dependent metabolic adaptations that support hepatocellular carcinoma growth and progression.

What methods should be employed to validate LYRM4 antibody specificity in experimental models?

Comprehensive validation of LYRM4 antibody specificity requires a multi-faceted approach combining molecular, cellular, and tissue-based techniques:

• Western blotting validation: Perform western blots on lysates from cells with known LYRM4 expression levels, including positive controls (LIHC cell lines), negative controls, and LYRM4 knockdown or knockout models. The antibody should detect a single band at the expected molecular weight (approximately 10 kDa for human LYRM4).

• Immunoprecipitation analysis: Use the LYRM4 antibody for immunoprecipitation followed by mass spectrometry to confirm that it specifically captures LYRM4 and its known binding partners in the NFS1-LYRM4-ACP complex.

• Immunohistochemistry with appropriate controls:

  • Positive tissue controls: LIHC tissues with confirmed LYRM4 overexpression

  • Negative tissue controls: Normal liver tissues with lower LYRM4 expression

  • Technical negative controls: PBS substitution for primary antibody

  • Peptide competition assays: Pre-incubation with the immunizing peptide should abolish specific staining

• Genetic validation: Compare staining patterns in tissues from wild-type versus LYRM4 knockdown/knockout models to confirm specificity.

• Cross-platform validation: Correlate LYRM4 protein detection by IHC with mRNA expression data from the same samples, similar to the approach used in studies correlating IHC findings with GEPIA2 database expression data .

• Subcellular localization assessment: Confirm that the staining pattern matches LYRM4's expected mitochondrial localization using confocal microscopy and co-localization with mitochondrial markers.

What is the optimal antibody-to-HRP ratio when preparing LYRM4-HRP conjugates?

The optimal antibody-to-HRP ratio for preparing LYRM4-HRP conjugates falls within specific parameters to ensure maximal conjugation efficiency while preserving antibody functionality. Based on molecular weight considerations (antibody ~160,000 Da versus HRP ~40,000 Da), the recommended molar ratios range between 1:4 and 1:1 antibody to HRP .

This translates to the following practical guidelines for different HRP quantities:

The lower end of the ratio range (1:4) generally produces conjugates with higher HRP density, potentially offering enhanced sensitivity but with a slightly increased risk of affecting antibody binding. The higher end of the ratio range (1:1) typically yields conjugates with preserved antibody binding characteristics but potentially lower enzymatic activity per antibody molecule .

For novel applications targeting LYRM4 in hepatocellular carcinoma tissue, preliminary optimization experiments comparing different ratios are recommended to determine the ideal balance between sensitivity and specificity for the particular experimental context.

How should researchers prepare tissue samples for optimal LYRM4 immunodetection?

Optimal LYRM4 immunodetection in hepatocellular carcinoma samples requires meticulous tissue preparation following a standardized protocol:

• Tissue collection and fixation:

  • Collect fresh tissue samples promptly after surgical resection

  • Fix immediately in 10% neutral-buffered formalin for 24-48 hours

  • Maintain a tissue-to-fixative ratio of at least 1:10 to ensure uniform fixation

• Tissue processing and embedding:

  • Dehydrate tissues through increasing ethanol gradients

  • Clear in xylene and infiltrate with paraffin

  • Embed in paraffin blocks with proper orientation to enable sectioning of representative areas

• Tissue microarray (TMA) construction:

  • Core selection: Select representative tumor and adjacent normal tissue areas based on H&E-stained sections

  • Core extraction: Use a tissue arrayer to extract 1-2mm cores from donor blocks

  • TMA assembly: Transfer cores to recipient blocks in an organized array format

• Sectioning and slide preparation:

  • Cut 4-5μm thick sections from paraffin blocks or TMAs

  • Mount on positively charged slides to enhance tissue adhesion

  • Dry slides overnight at 37°C to maximize tissue attachment

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Determine optimal retrieval conditions empirically for LYRM4 detection

  • Typically, pressure cooking for 3 minutes or water bath treatment at 95-98°C for 20-30 minutes

This standardized approach ensures consistent tissue quality and preservation of LYRM4 antigenicity, facilitating reliable immunohistochemical assessment across multiple specimens .

What experimental controls should be included when investigating LYRM4 expression in hepatocellular carcinoma?

A comprehensive control strategy is essential for reliable LYRM4 expression analysis in hepatocellular carcinoma research:

• Technical controls for immunohistochemistry:

  • Negative reagent control: Substitution of primary antibody with PBS to assess non-specific binding of detection reagents

  • Isotype control: Application of matched isotype antibody at the same concentration as LYRM4 antibody

  • Positive tissue control: Inclusion of a tissue with known LYRM4 expression in each staining batch

• Biological reference controls:

  • Paired adjacent normal tissue: Essential for evaluating LYRM4 overexpression in tumor context

  • Liver cirrhosis tissue: Important intermediate state to understand LYRM4 expression during hepatocarcinogenesis

  • Non-HBV related LIHC samples: To distinguish HBV-specific effects from general LIHC patterns

• Analytical validation controls:

  • Cross-platform validation: Correlation of IHC results with mRNA expression data from databases like GEPIA2

  • Multiple antibody validation: Use of different LYRM4 antibody clones targeting distinct epitopes

  • Protein expression quantification: Standardized scoring system with blinded assessment by multiple pathologists

• Functional controls:

  • Cell line panel: Analysis of LYRM4 expression across a spectrum of LIHC cell lines with varying aggressiveness

  • Enzyme activity assessment: Measurement of ISC-dependent proteins like mitochondrial complex I and ACO2 to correlate with LYRM4 expression levels

This multi-layered control strategy ensures robust and reproducible assessment of LYRM4 expression and its biological significance in hepatocellular carcinoma.

What quantification methods provide the most reliable assessment of LYRM4 expression in tissue samples?

Reliable quantification of LYRM4 expression in hepatocellular carcinoma tissue samples requires standardized, objective methodologies that minimize observer bias and maximize reproducibility:

• Immunohistochemistry scoring systems:

  • H-score method: Combines staining intensity (0-3) and percentage of positive cells (0-100%), yielding scores from 0-300

  • Allred score: Assesses proportion score (0-5) and intensity score (0-3), producing a total score of 0-8

  • Digital image analysis: Employs specialized software to quantify DAB staining intensity and distribution, providing continuous data on LYRM4 expression

• Semi-quantitative tissue microarray analysis:

  • Multiple cores per case (minimum 2-3) to account for tumor heterogeneity

  • Blinded evaluation by at least two independent pathologists

  • Consensus scoring for discrepant cases

• Multiplex quantification approaches:

  • Multiplex IHC: Simultaneous assessment of LYRM4 with other markers (e.g., immune cell markers)

  • Correlation analysis: Integration of LYRM4 expression with clinical parameters (age, serum LDL, TG)

• RNA-based quantification:

  • qRT-PCR: For LYRM4 mRNA quantification in fresh or frozen tissue

  • RNA-seq data analysis: Utilizing FPKM or TPM values from high-throughput sequencing

  • Database integration: Correlation with expression databases such as GEPIA2, LinkedOmics, and TIMER2.0

The combined implementation of protein-based and RNA-based quantification methods provides complementary data that strengthens the reliability of LYRM4 expression assessment, particularly when correlated with clinical outcomes.

What factors may compromise the stability of HRP-conjugated LYRM4 antibodies?

Multiple factors can compromise the stability of HRP-conjugated LYRM4 antibodies, requiring careful consideration during preparation, storage, and use:

• Buffer composition issues:

  • Presence of nucleophilic components like primary amines and thiols can react with LYNX chemicals, degrading conjugate quality

  • Sodium azide contamination irreversibly inhibits HRP activity, rendering the conjugate non-functional

  • Inappropriate buffer pH outside the 6.5-8.5 range may destabilize antibody structure or reduce conjugation efficiency

• Storage and handling challenges:

  • Repeated freeze-thaw cycles accelerate protein denaturation and enzymatic activity loss

  • Storage at inappropriate temperatures (room temperature or inconsistent refrigeration)

  • Exposure to strong light, particularly for fluorescence-tagged HRP conjugates

  • Bacterial contamination due to improper sterile technique

• Chemical degradation mechanisms:

  • Oxidative damage to both antibody and HRP components

  • Proteolytic degradation from trace contaminants

  • Aggregation during long-term storage

  • Loss of glycosylation on HRP affecting enzymatic activity

• Application-specific stressors:

  • Excessive dilution below effective concentration

  • Use with incompatible detection reagents

  • Extended incubation times at non-optimal temperatures

  • Repeated pipetting causing mechanical stress to the conjugate

To mitigate these stability challenges, researchers should prepare conjugates in recommended amine-free buffers, avoid sodium azide and nucleophilic components, store aliquots at -20°C for long-term storage or at 4°C for short-term use, and validate conjugate activity before critical experiments .

How can researchers minimize non-specific binding when using LYRM4 antibodies in hepatocellular carcinoma tissues?

Minimizing non-specific binding when using LYRM4 antibodies in hepatocellular carcinoma tissues requires a multi-faceted approach targeting various sources of background:

• Improved blocking protocols:

  • Use species-appropriate serum (5-10%) matching the secondary antibody host

  • Apply protein blockers like BSA (1-3%) or casein-based commercial blockers

  • Consider dual blocking with both serum and protein blockers

  • Extend blocking time to 60 minutes at room temperature for challenging tissues

• Antibody optimization:

  • Titrate primary LYRM4 antibody concentration carefully (typically starting at 1:100)

  • Perform antigen retrieval optimization specific to LYRM4 epitope accessibility

  • Increase washing duration and frequency between each step (minimum 3 x 5 minutes)

  • Incubate primary antibody at 4°C overnight rather than at higher temperatures

• Tissue-specific considerations:

  • Pre-treat sections with hydrogen peroxide (3%) to block endogenous peroxidase

  • Address liver-specific challenges such as high biotin content with avidin-biotin blocking kits

  • Consider using polymer-based detection systems instead of biotin-streptavidin for high-background tissues

  • Pre-absorb antibodies with liver tissue powder from normal liver if tissue-specific background persists

• Detection system modifications:

  • Use amplification systems with lower background characteristics

  • Reduce DAB development time with close monitoring for optimal signal-to-noise ratio

  • Consider alternative chromogens if DAB produces excessive background

  • Employ specialized detection kits designed for challenging tissue types

Implementation of these approaches should be systematic, changing one variable at a time while maintaining appropriate controls to identify the most effective combination for specific experimental conditions .

What strategies can resolve contradictory LYRM4 expression data between mRNA and protein levels?

Resolving contradictory LYRM4 expression data between mRNA and protein levels requires systematic investigation of biological and technical factors that may contribute to these discrepancies:

• Biological explanations assessment:

  • Post-transcriptional regulation: Evaluate microRNA targeting of LYRM4 transcripts using databases like LinkedOmics

  • Protein stability differences: Measure LYRM4 protein half-life in relevant cell models

  • Tissue heterogeneity: Compare microdissected tumor regions to bulk tissue analysis

  • Temporal dynamics: Consider time-course experiments to capture expression changes over time

• Technical validation approaches:

  • Multi-antibody confirmation: Utilize multiple LYRM4 antibodies targeting different epitopes

  • Cross-platform verification: Compare results across different mRNA quantification methods (qRT-PCR, RNA-seq, microarray)

  • Absolute quantification: Implement absolute mRNA (digital PCR) and protein (mass spectrometry) quantification

  • Sample preparation assessment: Evaluate the impact of different preservation methods on mRNA and protein detection

• Integrated analysis strategies:

  • Single-cell analysis: Apply single-cell RNA-seq and protein techniques to resolve cell-type specific expression patterns

  • Multi-omics correlation: Integrate transcriptomics, proteomics, and functional data from the same samples

  • Database triangulation: Compare findings with multiple databases (GEPIA2, LinkedOmics, TIMER2.0)

  • Clinical correlation: Determine whether mRNA or protein levels better correlate with clinical parameters and outcomes

• Functional validation:

  • LYRM4 knockdown/overexpression: Assess the functional consequences on ISC-dependent proteins and metabolic pathways

  • Enzymatic activity measurement: Correlate LYRM4 levels with activities of mitochondrial complex I and aconitate hydratase

  • Protein complex assessment: Evaluate the formation of the NFS1-LYRM4-ACP complex in relation to expression levels

This comprehensive approach helps distinguish genuine biological regulation from technical artifacts, providing a clearer understanding of LYRM4's expression patterns in hepatocellular carcinoma.

What approaches can overcome detection challenges for LYRM4 in highly fibrotic liver tissues?

Detecting LYRM4 in highly fibrotic liver tissues presents unique challenges that require specialized approaches to overcome background issues and epitope masking:

• Optimized antigen retrieval techniques:

  • Extended heat-induced epitope retrieval (HIER) with oscillating temperature cycles

  • Enzymatic retrieval with proteinase K titration for heavily cross-linked tissues

  • Combination approaches using both heat and enzymatic methods sequentially

  • Pressure cooker-based retrieval with optimized buffer systems for fibrous tissues

• Modified tissue preparation protocols:

  • Reduced fixation time for prospective samples to minimize protein cross-linking

  • Thinner tissue sections (2-3μm instead of standard 4-5μm) to improve reagent penetration

  • Extended deparaffinization and rehydration steps to ensure complete removal of paraffin

  • Pretreatment with permeabilization agents to enhance antibody access to heavily fibrotic regions

• Advanced detection strategies:

  • Tyramide signal amplification (TSA) to enhance detection sensitivity in challenging samples

  • Multi-round chromogenic staining with signal stripping between rounds

  • Fluorescence-based multiplex approaches with spectral unmixing to distinguish signal from autofluorescence

  • Digital enhancement and computational removal of collagen-associated background

• Specialized controls and validation:

  • Fibrosis-matched control tissues with known LYRM4 expression levels

  • Serial sections stained with collagen markers to correlate fibrotic areas with LYRM4 detection challenges

  • Cell-type specific markers to distinguish hepatocytes from activated stellate cells and fibroblasts

  • Laser capture microdissection of hepatocytes from fibrotic regions followed by PCR validation

These specialized approaches must be optimized for each specific research context, recognizing that fibrotic liver tissue presents variable challenges depending on etiology, duration, and severity of fibrosis. Systematic comparison of different detection methods with appropriate controls will identify the most effective approach for specific experimental conditions.