LYRM4 Antibody, FITC conjugated

What is LYRM4 and why is it important in cellular biology?

LYRM4 is a gene that encodes the Iron-Sulfur cluster biogenesis Desulfurase Interacting protein 11 kDa (ISD11), which plays a crucial role in the mitochondrial iron-sulfur cluster assembly pathway. The protein forms a complex with NFS1, a cysteine desulfurase that serves as a sulfur donor for iron-sulfur cluster synthesis. This interaction is essential as ISD11 stabilizes NFS1 and prevents its aggregation and proteolytic degradation . Iron-sulfur clusters are important prosthetic groups that define the functions of many proteins involved in vital cellular processes including oxidative phosphorylation, the citric acid cycle, iron homeostasis, heme biosynthesis, and DNA repair . Without properly functioning LYRM4/ISD11, iron-sulfur cluster biogenesis is compromised, potentially affecting multiple metabolic pathways.

What are the key applications for LYRM4 Antibody, FITC conjugated?

The LYRM4 Antibody, FITC conjugated, is suitable for multiple research applications including Western Blot (WB), Immunohistochemistry (IHC), Flow Cytometry (FC), and Immunocytochemistry/Immunofluorescence (ICC/IF) . In Western Blotting, it can be used at dilutions of 1:100-500, while for Immunohistochemistry, dilutions of 1:50-100 are recommended . For Flow Cytometry, the suggested dilution range is 1:10-50 . This versatility makes the antibody valuable for researchers investigating LYRM4 protein expression, localization, and interactions in various experimental contexts. The FITC conjugation eliminates the need for secondary antibody incubation, simplifying protocols and reducing background in fluorescence-based detection methods.

How specific is the LYRM4 Antibody, FITC conjugated for human samples?

The LYRM4 Antibody, FITC conjugated, has been specifically generated to recognize human LYRM4 protein. It is produced by immunizing rabbits with a KLH conjugated synthetic peptide corresponding to amino acids 46-73 from the central region of human LYRM4 . The antibody undergoes purification by Protein A affinity chromatography to ensure high specificity . According to the product information, it reacts with human samples but cross-reactivity with other species has not been extensively characterized in the provided search results . Researchers working with non-human samples should perform preliminary validation experiments to confirm antibody reactivity in their specific model organisms.

How can LYRM4 Antibody be used to investigate iron-sulfur cluster biogenesis disorders?

LYRM4 Antibody, FITC conjugated, provides a valuable tool for investigating disorders associated with defective iron-sulfur cluster biogenesis. Pathogenic mutations in LYRM4, such as the homozygous c.203G>T (p.R68L) mutation identified in patients with combined oxidative phosphorylation deficiency, can be studied using this antibody . Researchers can employ the antibody in immunohistochemistry or immunofluorescence to assess LYRM4 protein localization and expression levels in patient samples. Western blotting can be used to compare LYRM4 protein levels between control and patient samples, while flow cytometry enables quantitative analysis of LYRM4 expression in specific cell populations.

The antibody can also help investigate the downstream effects of LYRM4 mutations on iron-sulfur proteins within the respiratory chain complexes I, II, and III, all of which contain iron-sulfur clusters essential for their function . By correlating LYRM4 expression patterns with activities of these complexes, researchers can better understand the pathophysiological mechanisms of mitochondrial disorders involving iron-sulfur cluster biogenesis.

What methodological approaches can be used to study LYRM4/NFS1 complex formation using LYRM4 Antibody?

The interaction between LYRM4/ISD11 and NFS1 is crucial for iron-sulfur cluster biogenesis, and the LYRM4 Antibody, FITC conjugated, can be employed in several methodologies to study this complex:

• Co-immunoprecipitation followed by Western blotting: Researchers can use the antibody to pull down LYRM4 and its interacting partners (particularly NFS1) to analyze complex formation under various experimental conditions.

• Proximity ligation assay (PLA): The FITC-conjugated antibody can be combined with antibodies against NFS1 to visualize and quantify protein-protein interactions in situ.

• Immunofluorescence co-localization: The FITC-labeled LYRM4 antibody can be used alongside differently labeled antibodies against NFS1 to examine co-localization patterns in various subcellular compartments.

Experimental evidence indicates that mutations in LYRM4, such as the p.R68L variant, can affect the stability of the NFS1/ISD11 complex. In vitro studies showed that the NFS1Δ1-55/ISD11-R68L complex was more prone to aggregation compared to the wild-type complex . Additionally, the L-cysteine desulfurase activity of the mutant complex was substantially decreased compared to the wild-type, suggesting functional impairment . These methodological approaches using the LYRM4 antibody can help elucidate the structural and functional consequences of such mutations.

How can LYRM4 Antibody be utilized in cancer research, particularly for liver hepatocellular carcinoma (LIHC)?

Recent research indicates that LYRM4 is significantly upregulated in liver hepatocellular carcinoma (LIHC) at both mRNA and protein levels, suggesting its potential role as a prognostic biomarker . The LYRM4 Antibody, FITC conjugated, can be a valuable tool for researchers investigating this connection through:

• Immunohistochemical analysis of LIHC tissue microarrays: Researchers can use the antibody to examine LYRM4 expression patterns across different stages and grades of LIHC, correlating expression with clinical parameters such as age, serum low-density lipoprotein (LDL), and triglyceride (TG) content, which have shown significant associations with LYRM4 expression .

• Flow cytometric analysis of LIHC cell lines: The FITC-conjugated antibody enables quantitative assessment of LYRM4 expression levels in different hepatocellular carcinoma cell lines compared to normal hepatocytes.

• Investigation of LYRM4's role in ISC-dependent metabolic reprogramming: The antibody can help study how LYRM4 overexpression affects the activities of iron-sulfur proteins such as mitochondrial complex I and aconitase (ACO2), which show significantly increased activities in LIHC cell lines .

• Analysis of immune cell infiltration: LYRM4 expression in LIHC has been significantly positively correlated with infiltrating levels of six immune cell types, and both factors are strongly associated with prognosis . The antibody can be used in multicolor flow cytometry or immunofluorescence to study these relationships.

What are the optimal fixation and permeabilization conditions for LYRM4 immunofluorescence staining?

For optimal LYRM4 immunofluorescence staining using the FITC-conjugated antibody, consider the following protocol:

• Fixation: 4% paraformaldehyde in PBS for 15-20 minutes at room temperature is generally effective for preserving LYRM4 antigenic structure while maintaining cellular architecture. Since LYRM4/ISD11 is primarily localized in mitochondria (and to some extent in the nucleus), proper fixation is crucial for maintaining subcellular structures.

• Permeabilization: A gentle permeabilization with 0.1-0.3% Triton X-100 in PBS for 5-10 minutes is typically sufficient. For more delicate samples, 0.1-0.2% saponin can be used as an alternative.

• Blocking: Use 5% normal serum (from the same species as the secondary antibody would be, if not using a directly conjugated antibody) with 0.1% Triton X-100 and 1% BSA in PBS for 30-60 minutes to reduce nonspecific binding.

• Antibody dilution: For immunofluorescence applications, the LYRM4 antibody, FITC conjugated, should be used at approximately 1:50-1:100 dilution in blocking buffer .

• Counterstaining: Since LYRM4/ISD11 is predominantly mitochondrial, co-staining with mitochondrial markers such as MitoTracker or antibodies against COX IV can help confirm proper localization.

Note that optimization may be necessary depending on specific sample types, fixation sensitivity, and expression levels of LYRM4 in the target tissues.

What controls should be included when using LYRM4 Antibody, FITC conjugated, in experimental protocols?

To ensure reliable and interpretable results when using LYRM4 Antibody, FITC conjugated, researchers should include the following controls:

• Positive control: Include samples known to express LYRM4, such as HeLa cells, which have been documented to express LYRM4/ISD11 in both mitochondria and the nucleus . This confirms that the antibody and protocol are working properly.

• Negative control:

  • Isotype control: A FITC-conjugated rabbit polyclonal IgG that does not target any known antigens in your sample.

  • Samples with LYRM4 knockdown or knockout, if available.

  • Primary antibody omission control to assess background fluorescence.

• Specificity controls:

  • Pre-absorption control: Pre-incubate the antibody with excess recombinant LYRM4 protein or the immunizing peptide to confirm binding specificity.

  • Western blot validation showing a band at the expected molecular weight of LYRM4 (~11-12 kDa) to confirm antibody specificity before using in other applications.

• Technical controls:

  • For flow cytometry: Single-color controls for compensation settings when using multiple fluorophores.

  • For immunofluorescence: Autofluorescence control (sample with no antibody) to distinguish true signal from background.

Including these controls helps ensure that the observed staining patterns are specific to LYRM4 and not due to technical artifacts or non-specific binding.

How can LYRM4 Antibody be used in combination with functional assays to study iron-sulfur cluster biogenesis?

The LYRM4 Antibody, FITC conjugated, can be effectively integrated with functional assays to provide comprehensive insights into iron-sulfur cluster biogenesis:

What are common issues with LYRM4 Antibody in Western blotting and how can they be resolved?

When using LYRM4 Antibody, FITC conjugated, for Western blotting, researchers may encounter several challenges:

• Weak or absent signal:

  • Cause: Insufficient protein amount, antibody concentration too low, or protein degradation.

  • Solution: Increase protein loading (at least 20-30 μg of total protein), optimize antibody dilution (start with 1:100 as recommended ), and ensure proper sample preparation with protease inhibitors.

• Multiple bands or high molecular weight bands:

  • Cause: LYRM4/ISD11 can form complexes with NFS1 and other proteins that may not be fully denatured.

  • Solution: Ensure complete denaturation by increasing SDS concentration to 2% and boiling samples for 5-10 minutes. Consider using reducing agents like DTT or β-mercaptoethanol.

• High background with FITC-conjugated antibody:

  • Cause: Non-specific binding or excessive antibody concentration.

  • Solution: Increase blocking time (2 hours at room temperature or overnight at 4°C), use 5% BSA instead of milk for blocking, optimize antibody dilution, and increase washing steps (at least 3x15 minutes with 0.1% Tween-20 in PBS).

• Detection challenges with direct FITC conjugate:

  • Cause: FITC fluorescence may be less sensitive than chemiluminescence for Western blot detection.

  • Solution: Use a fluorescence-compatible imaging system with appropriate excitation (approximately 495 nm) and emission (approximately 520 nm) filters. Alternatively, consider using non-conjugated primary LYRM4 antibody with HRP-conjugated secondary antibody for enhanced sensitivity.

• Size discrepancy:

  • Cause: LYRM4/ISD11 has three known transcript variants , which may result in different protein sizes.

  • Solution: Reference the expected molecular weights for different isoforms and use positive controls with known expression patterns.

How can researchers optimize LYRM4 Antibody for flow cytometry applications?

To achieve optimal results when using LYRM4 Antibody, FITC conjugated, in flow cytometry:

• Sample preparation:

  • For intracellular staining of LYRM4, effective fixation and permeabilization are crucial since LYRM4/ISD11 is primarily localized in mitochondria.

  • Use 2-4% paraformaldehyde for fixation (10-15 minutes at room temperature) followed by permeabilization with 0.1% saponin or commercially available permeabilization buffers specifically designed for detecting intracellular/mitochondrial proteins.

• Antibody concentration:

  • Begin with the recommended dilution of 1:10-50 and titrate to determine optimal concentration.

  • Excessive antibody can increase background and reduce signal-to-noise ratio.

• Staining protocol:

  • Incubate cells with the antibody for 30-60 minutes at room temperature or 4°C in the dark to prevent photobleaching of the FITC fluorophore.

  • Include a protein-based blocking step (5-10% normal serum) before antibody addition to reduce non-specific binding.

  • Wash cells thoroughly (at least 2-3 times) after antibody incubation.

• Multicolor panel design:

  • When including LYRM4 Antibody, FITC conjugated, in multicolor panels:

    • Avoid fluorophores with spectral overlap with FITC (excitation maximum ~495 nm, emission maximum ~520 nm).

    • Include appropriate compensation controls.

    • Consider the relative abundance of LYRM4 – generally low to moderate expression in most cell types may require brighter fluorophores for detection.

• Data analysis considerations:

  • Use isotype controls to set negative gates appropriately.

  • Consider using median fluorescence intensity (MFI) rather than percent positive for quantitative comparisons of LYRM4 expression levels.

  • For mitochondrial proteins like LYRM4, correlate expression with mitochondrial mass using specific dyes like MitoTracker.

What approaches can be used to validate LYRM4 antibody specificity in research applications?

Validating the specificity of LYRM4 Antibody, FITC conjugated, is essential for generating reliable research data. Researchers should consider implementing these validation approaches:

• Genetic validation:

  • LYRM4 knockdown/knockout: Compare antibody staining in wild-type cells versus cells with reduced or absent LYRM4 expression (using siRNA, shRNA, or CRISPR-Cas9 technology).

  • Overexpression validation: Analyze increased signal in cells transfected with LYRM4 expression constructs.

• Biochemical validation:

  • Western blot analysis showing a band at the expected molecular weight of LYRM4 (~11-12 kDa).

  • Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (amino acids 46-73 of human LYRM4 ) before application to samples; specific staining should be abolished or significantly reduced.

• Orthogonal method validation:

  • Compare protein expression results obtained with the antibody to mRNA expression data from RT-PCR or RNA sequencing.

  • Use multiple antibodies targeting different epitopes of LYRM4 and compare staining patterns.

• Localization validation:

  • LYRM4/ISD11 has been reported to localize primarily to mitochondria, with some nuclear presence in HeLa cells .

  • Co-staining with established mitochondrial markers should show substantial co-localization.

  • Subcellular fractionation followed by Western blotting can confirm the expected distribution pattern.

• Cross-reactivity assessment:

  • Test the antibody against samples from different species if cross-species reactivity is claimed.

  • Examine staining in tissues known to have very low or no LYRM4 expression as negative controls.

How can LYRM4 Antibody contribute to understanding the role of iron-sulfur clusters in cancer metabolism?

The LYRM4 Antibody, FITC conjugated, offers valuable research opportunities for investigating the emerging role of iron-sulfur cluster biogenesis in cancer metabolism:

• Metabolic profiling combined with LYRM4 expression analysis:

  • Recent research has shown that the activities of ISC-dependent proteins such as mitochondrial complex I and aconitase (ACO2) are significantly increased in liver hepatocellular carcinoma (LIHC) cell lines compared to normal hepatocytes .

  • The FITC-conjugated LYRM4 antibody can be used to quantify LYRM4 expression levels in various cancer cell lines and patient samples via flow cytometry or immunohistochemistry.

  • Correlating LYRM4 expression with metabolic parameters (oxygen consumption rate, extracellular acidification rate) can reveal how iron-sulfur cluster biogenesis contributes to metabolic reprogramming in cancer.

• Investigation of iron-sulfur cluster-dependent DNA metabolism in cancer:

  • Several iron-sulfur proteins, including POLD1 and PRIM2, are involved in DNA replication and repair.

  • These proteins are significantly overexpressed in LIHC patients and correlated with poor prognosis .

  • Researchers can use the LYRM4 antibody alongside antibodies against these DNA metabolism proteins to investigate potential coordinated upregulation and functional relationships.

• Assessment of LYRM4's relationship with tumor immune microenvironment:

  • LYRM4 expression in LIHC has been significantly positively correlated with infiltrating levels of six immune cell types .

  • The FITC-conjugated antibody enables multicolor flow cytometry or immunofluorescence to analyze LYRM4 expression in relation to immune cell markers.

  • This approach could help elucidate the unexpected connection between iron-sulfur cluster biogenesis and tumor immunology.

• Therapeutic targeting potential:

  • As LYRM4 has been suggested as a potential molecular target for LIHC therapy , the antibody can be used to:

    • Screen for compounds that modulate LYRM4 expression or function

    • Evaluate the effects of candidate drugs on LYRM4 levels and localization

    • Serve as a companion diagnostic tool for stratifying patients who might benefit from therapies targeting iron-sulfur cluster biogenesis

What insights can LYRM4 Antibody provide about mitochondrial diseases involving iron-sulfur cluster deficiency?

The LYRM4 Antibody, FITC conjugated, represents a valuable tool for investigating mitochondrial diseases involving iron-sulfur cluster deficiency:

• Clinical sample analysis:

  • Mutations in LYRM4, such as the homozygous c.203G>T (p.R68L) mutation, have been identified in patients with combined oxidative phosphorylation deficiency affecting complexes I, II, and III .

  • Researchers can use the antibody to examine LYRM4 protein expression, stability, and localization in patient fibroblasts, muscle biopsies, or other available tissues.

  • Immunofluorescence microscopy with the FITC-conjugated antibody can reveal potential alterations in mitochondrial morphology or LYRM4 distribution in patient samples.

• Structure-function relationship studies:

  • The p.R68L mutation in LYRM4/ISD11 affects a highly conserved arginine residue .

  • Complementation studies in yeast showed that the equivalent mutation (p.R71L in yeast) resulted in decreased fitness .

  • The antibody can be used to study how this and other mutations affect LYRM4 protein stability, localization, and interactions with partners like NFS1.

• Tissue-specific effects of LYRM4 dysfunction:

  • The clinical presentation of LYRM4 mutations shows variable severity - from fatal neonatal disease to recovery and normal development .

  • The antibody enables comparative analysis of LYRM4 expression and localization across different tissues, potentially explaining the tissue-specific manifestations of iron-sulfur cluster biogenesis defects.

• Therapeutic monitoring:

  • The striking difference in clinical outcomes among patients with identical LYRM4 mutations suggests that environmental factors, such as the availability of the sulfur donor cysteine, might influence disease progression .

  • The antibody can be used to monitor changes in LYRM4 expression and function in response to potential therapeutic interventions, such as cysteine supplementation.

How can researchers integrate LYRM4 antibody data with systems biology approaches to understand iron-sulfur cluster biogenesis networks?

The integration of data generated using LYRM4 Antibody, FITC conjugated, with systems biology approaches offers powerful opportunities to comprehensively understand iron-sulfur cluster biogenesis networks: