LASP1 Antibody, FITC conjugated

What is LASP1 and what are its key structural and functional characteristics?

LASP1 (LIM and SH3 domain protein 1) was originally identified in metastatic breast cancer and functions primarily as a cytoskeleton protein. It contains an N-terminal LIM domain and two actin-binding domains in its core structure, with an SH3 domain at the C-terminus that enables interaction with proteins like zyxin . The gene is mapped to human chromosome 17q21 .

LASP1 plays crucial roles in dynamic actin-based cytoskeletal activities and is associated with the F-actin rich cortical cytoskeleton rather than actin stress fibers . It functions at cell membrane extensions and interacts with multiple proteins including F-actin, ANKRD54, and KBTBD10 . Additionally, LASP1 can transmit signals from the cytoplasm to the nucleus, contributing to its versatility in cellular processes .

The protein is expressed ubiquitously across normal tissues but with distinct expression patterns, and its molecular weight is calculated at approximately 30-35kD . Agonist-dependent changes in LASP1 phosphorylation may regulate actin-associated ion transport activities in parietal cells and other F-actin-rich secretory epithelial cell types .

What are the optimal fixation and permeabilization protocols for LASP1 immunofluorescence staining?

Based on established protocols for LASP1 antibody applications, the following methodology has proven effective:

Fixation Protocol:

• Fix samples with 4% paraformaldehyde for 10-15 minutes at room temperature or mild fixatives to preserve epitope accessibility.

• Wash three times with PBS containing 0.1% Triton X-100 and 1% DMSO (PBS-T) .

Permeabilization and Blocking:

• Permeabilize with PBS containing 0.1% Triton X-100 for 10 minutes at room temperature.

• Block non-specific binding using 10% normal goat serum in PBS-T containing 1% DMSO for 1 hour .

Primary Antibody Incubation:

• Incubate with rabbit anti-human LASP1 polyclonal antibody diluted 1:100 in blocking solution for 48 hours at 4°C .

Secondary Antibody Incubation:

• Wash three times with PBS-T containing 1% DMSO.

• Incubate with fluorescent secondary antibody (anti-rabbit conjugated with fluorophore) diluted 1:1000 in blocking solution overnight at 4°C or for 2 hours at room temperature .

Nuclear Counterstaining:

• Wash three times with PBS-T.

• Stain with DAPI (1:3000) for 10 minutes for nuclear visualization .

Mounting:

• Mount with appropriate mounting medium (e.g., Vectashield) .

This protocol has been validated in zebrafish embryonic tissue studies using light-sheet microscopy and fluorescence microscopy for LASP1 detection .

How do subcellular localization patterns of LASP1 differ across normal and cancerous tissues?

LASP1 exhibits distinctive subcellular localization patterns that correlate with physiological and pathological states:

Normal Tissues:

• Primarily cytoplasmic and cortical localization associated with the F-actin rich cytoskeleton

• Concentrated at sites of cell membrane extensions but absent from actin stress fibers

• Ubiquitous expression but with tissue-specific patterns

Cancer Tissues:

• High cytoplasmic expression often coupled with nuclear positivity in invasive breast carcinomas

• Significant correlation between nuclear LASP1 localization and tumor aggressiveness

• Progressive increase in expression from normal mucosa to colorectal cancer and metastatic colorectal cancer

The nuclear translocation of LASP1 in cancer cells represents a critical event in cancer progression. Research indicates that this nuclear presence correlates with poor patient outcomes and increased cellular proliferation . When designing LASP1 immunofluorescence experiments, researchers should employ z-stack imaging and high-resolution microscopy techniques to accurately distinguish between cytoplasmic, cortical, and nuclear LASP1 pools.

For quantitative assessment of LASP1 subcellular distribution, an immunoreactivity scoring system has been employed in breast cancer studies, showing significantly higher scores in invasive carcinomas compared to benign conditions such as mammary fibroadenomas .

What are the considerations for optimizing FITC-conjugated LASP1 antibody use in multi-parametric flow cytometry?

When incorporating FITC-conjugated LASP1 antibodies into multi-parametric flow cytometry panels, researchers should consider several technical aspects:

Spectral Considerations:

• FITC emits at approximately 519-525nm (green spectrum)

• Design panels to minimize spectral overlap with PE (yellow-orange) and other fluorophores with emission profiles close to FITC

Panel Design Strategy:

• Reserve FITC-LASP1 for intracellular detection while using alternative fluorophores for surface markers

• When studying LASP1 in combination with actin cytoskeleton components, consider:

  • Phalloidin-conjugates with far-red fluorophores for F-actin staining

  • Alternative fluorophores for vinculin, zyxin, or other cytoskeletal proteins

Fixation and Permeabilization Optimization:

• For intracellular LASP1 detection, validate multiple fixation/permeabilization combinations:

  • Formaldehyde fixation (2-4%) followed by saponin (0.1-0.5%) permeabilization

  • Methanol-based protocols for detection of LASP1 epitopes that might be masked by cross-linking fixatives

Controls Required:

• Fluorescence-minus-one (FMO) control to establish LASP1-negative gates

• Isotype control conjugated to FITC to assess non-specific binding

• Known LASP1-positive cell line (such as breast cancer cell lines) for positive control

• LASP1-knockdown samples as negative controls where available

Signal Amplification:
For detection of low-level LASP1 expression, consider a two-step approach using unconjugated primary anti-LASP1 followed by FITC-conjugated secondary antibody, which may provide stronger signal than direct FITC-conjugated LASP1 antibodies.

What controls should be incorporated when studying LASP1 expression and localization?

When designing experiments to investigate LASP1 expression and localization, incorporating appropriate controls is essential for reliable data interpretation:

Essential Controls for LASP1 Immunofluorescence:

Multi-channel Imaging Controls:

• Single-labeled samples for each fluorophore to establish bleed-through parameters

• Sequential image acquisition to minimize spectral overlap

• Z-stack imaging to accurately determine subcellular localization in three dimensions

Quantitative Analysis Controls:

• Include reference standards for signal intensity normalization across experiments

• Validate antibody linear detection range with titration experiments

• Include samples with known differential LASP1 expression (e.g., normal vs. cancer tissues) for assay validation

For studies examining LASP1's role in cancer progression, paired normal-tumor samples from the same patients should be analyzed to control for individual variation in expression patterns. This approach has been successfully employed in colorectal cancer studies investigating LASP1 as a potential biomarker .

How can LASP1 antibodies be effectively used to study its role in cancer metastasis?

LASP1 overexpression has been strongly linked to metastatic progression in multiple cancer types. To effectively study this relationship using LASP1 antibodies, researchers should consider the following comprehensive methodological approach:

Tissue Microarray Analysis:

• Apply standardized immunohistochemistry protocols using validated LASP1 antibodies

• Implement immunoreactivity scoring systems that have demonstrated clinical correlation in previous studies

• Analyze expression across primary tumors, lymph node metastases, and distant metastases

• Correlate LASP1 expression with established metastasis markers and patient outcomes

In Vitro Migration and Invasion Assays:

• Use FITC-conjugated LASP1 antibodies for live-cell tracking during migration

• Employ LASP1 immunofluorescence to examine localization at invasive structures:

  • Leading edge of migrating cells

  • Invadopodia formation sites

  • Cell-matrix adhesion complexes

Mechanistic Studies:

• Combine LASP1 antibody staining with actin cytoskeleton visualization to assess:

  • Co-localization with F-actin at membrane protrusions

  • Interaction with focal adhesion proteins

  • Changes in localization following hypoxia or growth factor stimulation

Animal Model Applications:

• Monitor LASP1 expression in primary tumors and metastatic sites using immunohistochemistry

• Correlate expression with tumor invasiveness and metastatic potential

• Assess effects of LASP1 knockdown on metastasis formation

This approach is supported by findings that LASP1 silencing significantly inhibits cell migration and invasion in multiple cancer cell lines, including pancreatic cancer (CFPAC-1, MIA-PaCa-2), hepatocellular carcinoma (HepG2, Huh-7), and esophageal squamous cell carcinoma (ECA109, KYSE510) . Conversely, LASP1 overexpression in cell lines with low endogenous LASP1 (BxPC-3, Panc-1) increases migration and invasion .

What are potential causes and solutions for discrepancies between LASP1 protein and mRNA expression levels?

Researchers often encounter discrepancies between LASP1 protein levels (detected by antibodies) and mRNA expression. Understanding and addressing these discrepancies requires consideration of several biological and technical factors:

Biological Factors:

Technical Considerations:

• Antibody Epitope Location:

  • Different antibodies may recognize distinct LASP1 domains

  • Solution: Use multiple antibodies targeting different LASP1 regions

• Extraction Efficiency:

  • Cytoskeletal-associated proteins like LASP1 may require specialized extraction protocols

  • Solution: Compare standard RIPA buffer with cytoskeletal extraction buffers

• Detection Method Sensitivity:

  • Western blotting vs. immunofluorescence sensitivity differences

  • Solution: Validate findings using multiple detection methods

• Reference Gene Selection:

  • Inappropriate housekeeping genes for mRNA normalization

  • Solution: Validate multiple reference genes for specific tissue/condition

The relationship between LASP1 mRNA and protein expression is particularly complex in cancer. For example, in pancreatic ductal adenocarcinoma, LASP1 expression is regulated by HIF1α through direct binding to a hypoxia response element in the LASP1 promoter . This mechanism explains how hypoxic conditions in the tumor microenvironment can drive LASP1 overexpression independent of gene amplification.

How can nuclear LASP1 localization be effectively detected and quantified?

Nuclear localization of LASP1 has significant implications for cancer progression and prognosis, but detecting and quantifying nuclear LASP1 presents several methodological challenges:

Optimized Nuclear LASP1 Detection Protocol:

• Fixation Considerations:

  • Use gentle crosslinking fixatives (2% paraformaldehyde for 10 minutes)

  • Avoid overfixation which can mask nuclear epitopes

  • Consider dual fixation approach: brief paraformaldehyde followed by methanol

• Nuclear Permeabilization:

  • Ensure complete nuclear membrane permeabilization with 0.5% Triton X-100

  • Increase permeabilization time to 15-20 minutes for dense tissues

• Antibody Selection:

  • Validate antibodies specifically for nuclear LASP1 detection

  • Consider using antibodies raised against different LASP1 epitopes

• Co-staining Approach:

  • Perform dual staining with DAPI for precise nuclear boundary determination

  • Consider co-staining with nuclear lamins to clearly delineate nuclear envelope

Quantification Strategies:

• Image Acquisition:

  • Collect z-stack images with minimal slice thickness (0.3-0.5μm)

  • Use confocal microscopy to accurately distinguish nuclear from perinuclear staining

• Analysis Methods:

  • Implement nuclear:cytoplasmic ratio calculations

  • Use automated image analysis with nuclear mask creation based on DAPI

  • Apply intensity thresholding based on negative controls

• Reporting:

  • Report percentage of cells with nuclear LASP1 positivity

  • Quantify mean nuclear LASP1 intensity relative to cytoplasmic intensity

  • Consider correlative analysis with markers of cell proliferation

In breast cancer studies, nuclear LASP1 localization has been successfully detected and correlated with aggressive disease characteristics . The nuclear translocation mechanism appears to be regulated by phosphorylation events and protein-protein interactions that may be cancer-type specific. Researchers should be aware that nuclear LASP1 may represent a distinct functional pool of the protein involved in transcriptional regulation, making its accurate detection particularly valuable for cancer progression studies.

How does LASP1 expression pattern differ across cancer types and what are the implications for using LASP1 antibodies as diagnostic tools?

LASP1 exhibits distinct expression patterns across various cancer types, with important implications for its potential as a diagnostic marker:

Cancer-Specific LASP1 Expression Patterns:

Diagnostic Application Considerations:

• Tissue-Based Diagnostics:

  • LASP1 immunohistochemistry provides post-surgical prognostic information

  • Most valuable when combined with other markers in multi-protein panels

  • Requires standardized scoring systems for clinical implementation

• Liquid Biopsy Applications:

  • Potential for urinary LASP1 detection in bladder cancer (TCC)

  • Limitations include false positives with urinary tract infections or hematuria (>250 erythrocytes/μl)

  • Requires specialized antibodies optimized for body fluid testing

• Methodological Requirements:

  • Antibody validation across multiple cancer types is essential

  • Establishment of cancer-specific expression thresholds

  • Development of automated quantification protocols

LASP1 has shown particular promise as a component of multi-gene/protein signatures. For example, it forms part of a six-gene signature predictive for disease progression and relapse in chronic myeloid leukemia . Additionally, in colorectal cancer, LASP1 has been identified as one protein in a panel with S100A9 and RhoGDI that can predict clinicopathological characteristics .

What considerations should be made when using LASP1 antibodies to study embryonic development?

LASP1 plays important roles in embryonic development, requiring specialized approaches when using LASP1 antibodies in developmental biology research:

Developmental Stage-Specific Considerations:

• Temporal Expression Analysis:

  • Conduct time-course studies at defined developmental stages

  • Zebrafish model enables LASP1 detection at 48 hpf, 72 hpf, and 6 days post-fertilization

  • Document stage-specific subcellular localization patterns

• Tissue Penetration Optimization:

  • Embryonic tissues often require extended antibody incubation times

  • For whole-mount embryos: primary antibody incubation for 48 hours at 4°C

  • Secondary antibody incubation overnight at 4°C

• Imaging Techniques:

  • 3D light-sheet microscopy enables whole-embryo visualization

  • Z-stack confocal imaging allows for tissue architecture preservation

  • Time-lapse imaging for dynamic LASP1 localization during development

Methodological Protocol for Embryonic LASP1 Detection:

• Embryo Preparation:

  • Fix embryos in 4% paraformaldehyde

  • Permeabilize with PBS containing 0.1% Triton X-100 and 1% DMSO

  • Block with 10% normal goat serum in PBS-T with 1% DMSO

• Antibody Application:

  • Use rabbit anti-human LASP1 polyclonal antibody (1:100 dilution)

  • Incubate for 48 hours at 4°C

  • Apply fluorescent secondary antibody (e.g., anti-rabbit Alexa 488, 1:1000) overnight

• Mounting for Imaging:

  • Place embryos in 0.8% low-melting agarose

  • Position in instrument holder for optimal visualization

• Controls:

  • Include developmental stage-matched negative controls

  • Process without primary antibody to assess background fluorescence

This approach has been successfully implemented in zebrafish models to characterize LASP1 expression during embryonic development, revealing stage-specific and organ-specific expression patterns . When studying development, researchers should be particularly attentive to background fluorescence, as embryonic tissues can exhibit high levels of autofluorescence that may interfere with specific LASP1 detection.

What emerging technologies could enhance the detection and functional analysis of LASP1?

Several cutting-edge technologies show promise for advancing LASP1 research beyond traditional antibody-based methods:

Advanced Imaging Technologies:

• Super-Resolution Microscopy:

  • Stimulated Emission Depletion (STED) microscopy for resolving LASP1 localization at actin-rich structures

  • Single-molecule localization microscopy (PALM/STORM) to map precise LASP1 distribution patterns at nanometer resolution

  • Expansion microscopy to physically enlarge specimens for enhanced visualization of LASP1-cytoskeletal interactions

• Intravital Imaging:

  • Monitor LASP1 dynamics in living organisms using fluorescently-tagged LASP1

  • Track cancer cell invasion and metastasis with LASP1 as a marker in real-time

  • Assess therapeutic response through LASP1 expression and localization changes

Molecular Interaction Analysis:

• Proximity Ligation Assay (PLA):

  • Detect LASP1 interactions with binding partners in situ

  • Quantify dynamic changes in interaction networks under different conditions

  • Identify novel LASP1 interactors in different subcellular compartments

• Mass Spectrometry-Based Proteomics:

  • Phospho-proteomics to map phosphorylation sites affecting LASP1 function

  • Interaction proteomics to identify cancer-specific LASP1 binding partners

  • Spatial proteomics to determine precise subcellular distribution patterns

Functional Analysis Tools:

• CRISPR-Cas9 Gene Editing:

  • Generate domain-specific LASP1 mutants to dissect functional regions

  • Create cell and animal models with fluorescent protein-tagged endogenous LASP1

  • Develop conditional knockout systems for tissue-specific LASP1 ablation

• Single-Cell Analysis:

  • Single-cell RNA-seq combined with protein detection to correlate LASP1 mRNA and protein levels

  • Mass cytometry (CyTOF) for multi-parameter analysis of LASP1 in relation to other cancer markers

  • Spatial transcriptomics to map LASP1 expression in tissue context

These emerging technologies will enable researchers to move beyond simple detection of LASP1 toward deeper understanding of its dynamic regulation and functional significance in both normal development and cancer progression. Integration of multiple approaches will be particularly valuable, as LASP1's role at the intersection of structural and signaling functions requires sophisticated analysis methods.

How can contradictory findings about LASP1 function be reconciled through improved antibody-based methodologies?

The literature contains some contradictory findings regarding LASP1 function and expression patterns. Improved antibody-based methodologies can help resolve these discrepancies:

Sources of Contradictory Results:

• Gene Amplification vs. Transcriptional Regulation:

  • Early studies suggested LASP1 overexpression in breast cancer was due to gene amplification

  • Later research demonstrated that overexpression often occurs without gene amplification through transcriptional mechanisms like HIF1α regulation

• Prognostic Significance Variations:

  • Some studies reported LASP1 overexpression correlates with poor prognosis

  • Contradictory findings showed LASP1 underexpression in patients who died within 5 years after surgery compared to long-term survivors

Methodological Solutions:

• Antibody Standardization:

  • Use antibodies targeting conserved LASP1 epitopes across studies

  • Implement rigorous validation protocols including Western blot, immunoprecipitation, and knockdown controls

  • Develop reference standards for antibody performance evaluation

• Context-Specific Analysis:

  • Account for tissue-specific LASP1 isoforms and post-translational modifications

  • Analyze LASP1 in context of upstream regulators (e.g., HIF1α) and downstream effectors

  • Consider cell type-specific functions when interpreting results

• Comprehensive Detection Approach:

  • Employ multi-epitope detection with antibodies recognizing different LASP1 domains

  • Combine protein and mRNA detection in the same samples

  • Implement quantitative scoring systems with clear thresholds for positivity

• Experimental Design Improvements:

  • Include larger, more homogeneous patient cohorts

  • Stratify analyses by cancer subtype, stage, and treatment history

  • Use paired normal-tumor samples to control for individual variation

A notable example of reconciling contradictory findings comes from breast cancer studies, where initial reports of LASP1 overexpression being mediated by gene amplification were later challenged. More thorough analyses using laser-capture microdissected breast cancer cells and immunohistochemistry demonstrated that LASP1 overexpression is not necessarily correlated with copy number gains . This highlights the importance of using multiple methodological approaches and carefully controlled experiments to resolve discrepancies in LASP1 research.