LZTS1 Antibody, HRP conjugated

What epitope regions are targeted by available LZTS1 antibodies and how does this affect experimental design?

Commercial LZTS1 antibodies target various epitope regions including amino acids 400-550 , 514-596 , and 47-212. When designing experiments, researchers should select antibodies targeting regions that: (1) avoid splice variant regions if studying all LZTS1 isoforms, (2) contain the specific domain of interest for functional studies, or (3) expose reliably in the fixed/processed state of your samples. For example, antibodies targeting the AA 400-550 region have been validated for IHC-P, WB, and ICC/IF applications with human samples , while those targeting AA 514-596 have been specifically validated for Western blotting and ELISA .

What are the optimal dilution ratios for LZTS1 antibodies across different applications?

Based on validated protocols, recommended dilutions vary by application:

• Western Blotting: 1:1000 for polyclonal LZTS1 antibody (e.g., GTX117376) or 0.4 μg/mL (e.g., ab251681)

• Immunohistochemistry (IHC-P): 1:200 dilution for paraffin-embedded tissues

• Immunocytochemistry/Immunofluorescence (ICC/IF): 4 μg/ml for cell staining

• ELISA: Concentration depends on specificity requirements and signal-to-noise ratio optimization

Each application requires distinct optimization strategies. For HRP-conjugated antibodies specifically, initial titration experiments comparing 1:500, 1:1000, and 1:2000 dilutions are recommended to determine optimal signal-to-noise ratios while minimizing background.

What fixation and antigen retrieval methods optimize LZTS1 detection in tissue and cellular samples?

For optimal LZTS1 immunodetection:

• Cellular samples: PFA-fixation (4% paraformaldehyde) followed by Triton X-100 permeabilization has been validated for SK-MEL-30 cells

• Tissue samples: For paraffin sections, citrate buffer (pH 6.0) heat-induced epitope retrieval for 20 minutes has shown efficacy with human pancreas tissue

• Electron microscopy: Gold-particle immunolabeling techniques can detect LZTS1 association with adherens junction (AJ) belts and intracellular distributions

The subcellular localization pattern varies, with some cells showing apical endfeet localization in a ring-like pattern and co-localization with ZO1 (a scaffolding protein in adherens junctions) .

How should I validate the specificity of HRP-conjugated LZTS1 antibodies?

Multiple validation strategies should be employed:

• Positive and negative control tissues/cells (LZTS1 is expressed in subsets of aRGs, IPs, and neurons )

• Western blot analysis showing expected band size (compare with vector-only transfected HEK-293T lysate as control )

• Peptide competition assay using the immunogen peptide to confirm specificity

• Comparison with unconjugated primary LZTS1 antibody plus HRP-secondary antibody detection

• Knockdown/knockout validation in cells with confirmed LZTS1 expression

• Cross-reference with RNA expression data (in situ hybridization or transcriptomics)

How can LZTS1 antibodies be incorporated into multi-parameter flow cytometry panels?

For multi-parameter flow cytometry incorporating LZTS1:

• Fixation and permeabilization: Use commercial intracellular staining kits compatible with transcription factor detection

• Panel design: Pair HRP-conjugated LZTS1 antibody with fluorochrome-conjugated surface markers using fluorochromes with minimal spectral overlap

• Intracellular marker timing: Add LZTS1 antibody after surface marker staining and fixation/permeabilization

• Signal development: For HRP-conjugated antibodies, use tyramide signal amplification (TSA) with fluorescent substrates like Tyramide-Alexa Fluor conjugates

• Controls: Include FMO (fluorescence minus one) controls and single-stained compensation controls

This approach enables correlation of LZTS1 expression with cell cycle status by co-staining with DNA content markers and proliferation markers like Ki-67.

What experimental approaches can resolve the contradictory tumor suppressor versus oncogenic roles of LZTS1 in different cancer types?

This contradiction requires systematic experimental approaches:

• Tissue-specific conditional knockout/knockin models to evaluate cancer development rates

• ChIP-seq analysis to identify direct transcriptional targets and regulatory mechanisms

• Proximity labeling (BioID/APEX) to map tissue-specific protein interaction networks

• Comparison of LZTS1 post-translational modifications across cancer types using phospho-specific antibodies

• Integrated multi-omics analysis correlating LZTS1 expression with PI3K-AKT pathway activation and EMT markers (N-cadherin, E-cadherin)

• Cell-type specific LZTS1 overexpression/knockdown followed by phenotypic assays (proliferation, migration, invasion)

These approaches help determine whether context-dependent factors influence LZTS1 function, potentially explaining its dual role as both tumor suppressor and oncogene.

How can HRP-conjugated LZTS1 antibodies be utilized for chromatin immunoprecipitation (ChIP) experiments?

While HRP-conjugated antibodies are not typically used for ChIP, an adapted protocol could include:

• Chromatin preparation: Standard cross-linking with formaldehyde and sonication to 200-500bp fragments

• Pre-clearing: Incubate chromatin with protein A/G beads to reduce background

• Immunoprecipitation: Use HRP-conjugated LZTS1 antibody with anti-HRP antibody-coated magnetic beads

• Sequential ChIP: For co-occupancy studies with transcription factors interacting with LZTS1

• Library preparation: Standard ChIP-seq library preparation following elution and cross-link reversal

• Data analysis: Peak calling and motif analysis to identify genomic binding regions

This approach requires rigorous optimization and validation compared to conventional ChIP antibodies.

What strategies can address inconsistent LZTS1 detection across different sample types?

Inconsistent LZTS1 detection may stem from:

• Sample-specific issues: Optimize protein extraction buffers (add phosphatase inhibitors for phosphorylated forms)

• Expression levels: Increase antibody concentration or extend incubation time for low-expressing samples

• Epitope accessibility: Test multiple antibodies targeting different regions (N-terminal vs. C-terminal)

• Post-translational modifications: Use phospho-specific antibodies if phosphorylation affects epitope recognition

• Fixation artifacts: Compare multiple fixation methods (PFA vs. methanol)

• Heterogeneous expression: LZTS1 shows cell-type specific expression patterns with highest levels in differentiating neural cells

Compare results with RNA expression data to determine if inconsistencies reflect biological variation or technical limitations.

How should researchers interpret contradictory results between LZTS1 expression and functional outcomes in different cancer models?

Contradictory results require systematic analysis:

• Cell-type specificity: LZTS1 function may depend on cellular context and existing signaling networks

• Isoform analysis: Verify whether different splice variants are being detected across studies

• Post-translational modifications: Phosphorylation status may alter function without changing expression levels

• Pathway interactions: Analyze PI3K-AKT pathway status and EMT marker expression in each model

• Genetic background: Consider how model-specific genetic alterations influence LZTS1 functionality

• Temporal dynamics: Early tumor suppressive effects might contrast with later oncogenic roles during disease progression

These factors help reconcile apparently contradictory findings that LZTS1 functions as a tumor suppressor in some contexts but promotes tumorigenesis in colorectal cancer models .

What controls are essential when using HRP-conjugated LZTS1 antibodies to ensure signal specificity?

Essential controls include:

• Substrate-only control: Eliminates possibility of endogenous peroxidase activity

• Isotype-matched, HRP-conjugated control antibody: Detects non-specific binding

• Blocking peptide competition: Confirms signal specificity to the target epitope

• Known positive and negative tissue/cell controls: Validates staining pattern

• Comparison with independent detection methods: RNA in situ hybridization or unconjugated primary + secondary detection

• Serial dilution validation: Confirms signal diminishes proportionally with antibody dilution

For Western blotting specifically, include vector-only transfected HEK-293T lysate as a negative control alongside LZTS1-expressing samples .

How does LZTS1 contribute to neuronal delamination and what techniques can investigate this function?

LZTS1 plays crucial roles in neuronal delamination processes:

• It localizes to adherens junctions at the apical endfeet of neural progenitor cells in a ring-like pattern

• Co-localizes with ZO1, a scaffolding protein in adherens junctions

• Shows association with adherens junction belts as demonstrated by electron microscopy

• Is expressed in nascent differentiating cells, including those positive for Tbr2(Eomes)::EGFP and Gadd45g::d4Venus

Investigation techniques include:

• Live imaging with fluorescently tagged LZTS1 to track delamination dynamics

• Proximity labeling to identify interaction partners at adherens junctions

• Conditional knockout models to assess cortical development

• Electron microscopy with immunogold labeling to precisely localize LZTS1 at subcellular structures

• In utero electroporation for acute manipulation of LZTS1 expression in developing brain

What methodological approaches can determine how LZTS1 influences the PI3K-AKT pathway and EMT in colorectal cancer?

Recent findings suggest LZTS1 promotes tumorigenesis through AKT activation and EMT . To investigate:

• Phospho-protein analysis: Quantify phosphorylated AKT, GSK3β, and other pathway components in LZTS1-manipulated cells

• Protein-protein interaction studies: Co-immunoprecipitation to identify direct interactions with PI3K components

• Transcriptional profiling: RNA-seq following LZTS1 overexpression/knockdown to identify downstream transcriptional changes

• EMT marker analysis: Quantify E-cadherin, N-cadherin, vimentin, and other EMT markers by immunoblotting and immunofluorescence

• Migration/invasion assays: Assess functional consequences of LZTS1 manipulation on cell motility

• In vivo models: Xenograft studies with LZTS1-manipulated cells to evaluate tumor growth and metastatic potential

These approaches systematically evaluate the mechanistic connection between LZTS1 expression and oncogenic processes in colorectal cancer .

How can researchers integrate findings on LZTS1's dual roles in cell cycle regulation and cytoskeletal organization?

LZTS1's involvement in both cell cycle (through CDC2-cyclin B1 stabilization) and cytoskeletal regulation (through adherens junction association) suggests integrated cellular functions. Research approaches include:

• Super-resolution microscopy to visualize LZTS1 localization during different cell cycle phases

• BioID proximity labeling at different cell cycle stages to identify phase-specific interaction partners

• Phospho-proteomics to map LZTS1 phosphorylation dynamics throughout the cell cycle

• Domain-specific mutations to separate cytoskeletal vs. cell cycle regulatory functions

• Correlation analysis between LZTS1 expression and cell cycle/cytoskeletal genes across large cancer datasets

• Live-cell imaging with fluorescently tagged LZTS1 and cytoskeletal/cell cycle markers