LANCL1 Antibody

What is LANCL1 and why is it significant in research?

LANCL1 (LanC lantibiotic synthetase component C-like 1, also known as P40 or GRP69A) is a 399-amino acid protein (45 kDa calculated, 40 kDa observed) homologous to bacterial lanthionine synthetase C (LanC) family. It functions as a glutathione transferase and plays critical roles in:

• Cellular antioxidant defense mechanisms

• Redox homeostasis regulation

• Glutathione (GSH) binding and metabolism

• Neuroprotection against oxidative stress

• Tumor initiation processes in certain cancers

LANCL1 is primarily expressed in neural tissues and testis, with emerging roles in multiple pathologies including hepatocellular carcinoma, neuropathic pain, amyotrophic lateral sclerosis, and prostate cancer .

What factors should be considered when selecting a LANCL1 antibody for research applications?

When selecting a LANCL1 antibody, researchers should consider:

For studying LANCL1's role as a cell surface protein in HCC, antibodies targeting the extracellular N-terminal domain have demonstrated functional inhibition of sphere-forming ability .

What are the recommended protocols for Western blot detection of LANCL1?

For optimal Western blot detection of LANCL1:

Sample Preparation:

• For tissue samples: Use RIPA buffer with protease inhibitors

• For brain/testis tissue: These provide strong positive controls

• Expected band: ~40 kDa

Protocol Recommendations:

• Use 10% SDS-PAGE for optimal separation

• Transfer to PVDF membranes (recommended over nitrocellulose)

• Block with 3% BSA in PBS (preferable to milk for this target)

• Primary antibody dilutions:

  • Polyclonal: 1:500-1:1000 (e.g., 12647-1-AP)

  • Monoclonal: 1:5000-1:50000 (e.g., 68160-1-Ig)

• Protein loading controls: β-actin works well with LANCL1 detection

Validation Controls:

• Positive tissue controls: Mouse brain, testis, HeLa cells, HepG2 cells

• Negative controls: LANCL1 knockdown cells have been validated

• Cross-reactivity: Minimal issues reported with specific antibodies

How can I effectively perform immunofluorescence staining for LANCL1?

For successful immunofluorescence detection of LANCL1:

Cell Preparation:

• U-251 cells have been validated for positive IF/ICC detection

• Both permeabilizing and non-permeabilizing conditions may be used depending on the research question

Protocol Optimization:

• Fixation: 4% paraformaldehyde (10 minutes) generally works well

• For membrane localization studies: Include both permeabilized and non-permeabilized samples

• Antigen retrieval: Not typically required for cultured cells

• Primary antibody dilutions:

  • Polyclonal: 1:50-1:500 (e.g., 12647-1-AP)

  • Monoclonal: 1:400-1:1600 (e.g., 68160-1-Ig)

• Counter-staining: DAPI (1:1000) works well for nuclear visualization

Applications:

• Particularly useful for studying:

  • Subcellular localization (cytoplasmic and membranous patterns)

  • Co-localization with binding partners (e.g., FAM49B, CBS)

  • Cell surface expression in non-permeabilized conditions

What approaches can be used to verify LANCL1 knockdown or overexpression models?

To establish and validate LANCL1 manipulation models:

Knockdown Verification:

• RT-qPCR: Using validated primers (e.g., forward: 5′-TTGCCTTGCTTTTCGCTGAC-3′; reverse: 5′-GGCAGTCACATCCCATTCCA-3′)

• Western blot: Compare protein levels to control cells

• Functional validation: Assess phenotypic changes related to LANCL1 function

Overexpression Confirmation:

• Western blot: Detect increased LANCL1 expression

• Flow cytometry: For cell surface LANCL1 quantification

• Tagged constructs: Use anti-tag antibodies (e.g., FLAG, YFP) for detection

• Rescue experiments: Restore LANCL1 function with shRNA-resistant constructs

Control Considerations:

• Include appropriate vector controls

• Use shRNA-resistant LANCL1 variants for rescue experiments

• Perform multiple knockdown approaches (siRNA and shRNA) to confirm specificity

• For structure-function studies, confirm expression of mutant variants

How can I investigate LANCL1's role in protein-protein interactions within my experimental system?

To study LANCL1's interactions with partner proteins:

Co-immunoprecipitation (Co-IP):

• Use 0.5-4.0 μg anti-LANCL1 antibody (e.g., 68160-1-Ig) per 1.0-3.0 mg total protein lysate

• Protein G purification is recommended for monoclonal antibodies

• Validate with reverse Co-IP using antibodies against suspected interaction partners

• Examples of validated interactions include FAM49B, CBS, and TRIM21

Proximity Ligation Assays:

• Useful for detecting in situ protein interactions with spatial resolution

• Requires antibodies raised in different host species

Mass Spectrometry Approaches:

• Immunoprecipitate LANCL1 complexes

• Follow with LC-MS/MS analysis

• Validate top hits with orthogonal methods (Co-IP, functional studies)

Functional Validation:

• Use domain mapping to identify interaction interfaces

• Employ mutational analysis to disrupt specific interactions

• Assess downstream functional consequences of disrupted interactions

When studying LANCL1-FAM49B interaction, consider investigating the role of TRIM21 in regulating this complex through ubiquitin-proteasome degradation .

What methodologies are recommended for studying LANCL1's role in redox regulation and oxidative stress?

For investigating LANCL1's functions in redox homeostasis:

ROS Detection Methods:

• DCF-DA fluorescence assay for intracellular ROS quantification

• Dihydroethidium (DHE) for superoxide detection

• MitoSOX for mitochondrial superoxide measurement

• Compare LANCL1-KD, wild-type, and rescue models

Glutathione Assays:

• Measure GSH/GSSG ratios as LANCL1 is a GSH-binding protein

• Assess glutathione transferase activity with artificial substrates like CDNB

Transcriptomic Analysis:

• RNA-seq comparing control, LANCL1-KD, and LANCL1-rescue samples

• Focus on ROS-responsive gene sets (e.g., Hallmark Reactive Oxygen Species Pathways)

Pathway Analysis:

• Investigate LANCL1's regulation of the Rac1-NADPH oxidase pathway

• Examine independence from glutathione transferase function

• Assess activation of antioxidant response elements and NRF2 signaling

In vivo Models:

• Measure oxidative damage markers in LANCL1-KO vs. wild-type mice

• Compare across tissues with differential LANCL1 expression

How can LANCL1 antibodies be used to examine its involvement in cancer pathogenesis?

For studying LANCL1's role in cancer biology:

Tumor Initiation Studies:

• Sphere formation assays to assess cancer stem cell-like properties

• Limiting dilution assays in vivo for tumor-initiating ability

• Use anti-LANCL1 antibodies (particularly targeting N-terminal region) as functional blockers

Expression Profiling:

• IHC staining of tumor tissue microarrays

• Correlation with clinical parameters (stage, survival)

• Co-expression analysis (e.g., LANCL1 and FAM49B)

Mechanistic Investigations:

• Analyze LANCL1's effect on ROS levels in tumor spheroids and xenografts

• Examine signaling pathway modulation (FAM49B-Rac1-NADPH oxidase axis)

• Study chemoresistance mechanisms in LANCL1-high vs. LANCL1-low cells

Therapeutic Targeting:

• Blocking antibody approaches targeting cell surface LANCL1

• Combination with ROS-inducing therapies

• Assessment of specificity using LANCL1-KD controls

High co-expression of LANCL1 and FAM49B has been associated with more advanced tumor stage and poorer survival in HCC patients .

What are the common technical challenges when working with LANCL1 antibodies and how can they be addressed?

Sample-dependent optimization is often necessary, especially when transitioning between different experimental systems .

How can I differentiate between LANCL1 and other LANCL family members in my experiments?

To ensure specificity for LANCL1 over other family members (LANCL2, LANCL3):

Antibody Selection:

• Verify epitope mapping to ensure targeting LANCL1-specific regions

• Review cross-reactivity data in validation studies

• Consider antibodies raised against unique N-terminal regions

Validation Approaches:

• Western blot analysis in tissues with differential expression

  • LANCL1: Highly expressed in brain and testis

  • LANCL2: More broadly expressed across tissues

  • LANCL3: Lower expression levels in most tissues

• Include LANCL1-specific knockdown/knockout controls

• Parallel detection with isoform-specific antibodies

Molecular Analyses:

• Use isoform-specific primers for qPCR validation

• LANCL1 primers: forward: 5′-TTGCCTTGCTTTTCGCTGAC-3′; reverse: 5′-GGCAGTCACATCCCATTCCA-3′

• Consider unique restriction sites for confirmation

Functional Differentiation:

• LANCL1: GSH binding, antioxidant defense, CBS inhibition

• LANCL2: ABA binding, glucose transport regulation

• Perform function-specific assays to confirm identity

How can LANCL1 antibodies be employed to investigate its role in neurodegenerative diseases?

For studying LANCL1 in neurodegeneration:

Translational Applications:

• Immunohistochemical analysis of brain tissues from neurodegenerative disease models

• Co-localization studies with neurodegeneration markers

• Assessment of LANCL1 expression changes during disease progression

ALS Research Applications:

• Compare LANCL1 levels in motor neurons of ALS models vs. controls

• Investigate LANCL1-mediated regulation of AKT activity

• Use LANCL1 antibodies to track transgene expression in CNS-specific LANCL1 overexpression models

Neuropathic Pain Studies:

• Employ LANCL1 as a key immune marker in neuropathic pain models

• Explore the miR-6325/LANCL1 axis in pain development

• ROC analysis showed LANCL1 has predictive value for neuropathic pain (AUC = 0.870 in GSE70006; AUC = 0.806 in GSE91396)

Methodological Approach:

• IHC with anti-LANCL1 antibody (1:50-1:500 dilution)

• DAPI counterstaining for nuclei identification

• Quantification of neuronal LANCL1 expression in affected vs. unaffected regions

• Correlate with markers of oxidative stress and neuroinflammation

What novel approaches can be used to study LANCL1 as a cell surface protein in therapeutic development?

For investigating LANCL1's potential as a therapeutic target:

Cell Surface Localization Studies:

• Flow cytometry using anti-LANCL1 antibodies in non-permeabilized cells

• Compare signal in permeabilized vs. non-permeabilized conditions

• Use antibodies targeting the N-terminal domain (residues 1-47)

• Validate with confocal microscopy and membrane fractionation

Antibody-Based Therapeutic Approaches:

• Anti-LANCL1 antibodies targeting the extracellular N-terminal domain have shown inhibition of sphere formation in HCC cells

• Test combination treatments with standard chemotherapies

• Evaluate antibody penetration in spheroid models

Structure-Function Analysis:

• Generate Flag-tagged LANCL1 variants with mutations in key domains

• Assess cell surface localization by immunofluorescence

• Correlate surface expression with functional outcomes

Experimental Design:

• Express wild-type and mutants of LANCL1 with Flag-tag at different positions

• Perform immunofluorescence using anti-Flag antibody under both permeabilizing and non-permeabilizing conditions

• Quantify surface expression by flow cytometry

• Correlate with tumor-initiating abilities in functional assays

What methodological approaches are recommended for investigating LANCL1's role in reproductive biology?

For studying LANCL1 in reproductive tissues and functions:

Expression Analysis in Testis:

• IHC staining of testicular tissue sections

• Stage-specific expression during spermatogenesis

• qPCR analysis using validated primers: forward: 5′-TTGCCTTGCTTTTCGCTGAC-3′; reverse: 5′-GGCAGTCACATCCCATTCCA-3′

• Western blot detection (expect 40 kDa band)

Transgenic Model Analysis:

• Compare wild-type, heterozygous (LanCL1+/−), and homozygous knockout (LanCL1−/−) mice

• Assess reproductive capacity, sperm motility, and testicular development

• CRISPR/Cas9 technology has been used to generate such models

Single-Cell RNA-Seq Integration:

• Analyze LANCL1 expression in spermatocytes and spermatids

• Investigate effects on germ cells and stromal cells

• Explore pathways affected (P53 signaling, PPAR signaling)

• Examine relationship with M2 macrophage polarization

Functional Assessment:

• Mouse breeding experiments to evaluate fertility

• Semen analysis for sperm parameters

• Immunofluorescence to locate LANCL1 in reproductive tissues

• Hormone analysis including testosterone measurement by ELISA

Knockout studies have shown that the LANCL1 gene significantly influences reproduction ability and sperm motility in male mice, though no effects on testosterone synthesis were observed .