LAP1 Antibody

What is LAP1 and why is it significant in biological research?

LAP1 is an integral protein of the inner nuclear membrane that is ubiquitously expressed throughout tissues. It plays a critical role in maintaining nuclear envelope architecture through binding to lamins and chromatin . The significance of LAP1 extends to its interactions with other proteins, notably torsinA and emerin, which are implicated in DYT1 dystonia and X-linked Emery-Dreifuss muscular dystrophy, respectively . Recent research has also revealed LAP1's role in cancer progression, particularly in melanoma, where it contributes to nuclear adaptability during cell migration and invasion . These multifaceted functions make LAP1 an important target for research across various biological disciplines including cell biology, oncology, and neurology.

What are the known isoforms of human LAP1 and how can they be distinguished experimentally?

Human cells express two main LAP1 isoforms: LAP1B and LAP1C. These isoforms differ in their amino terminus, with LAP1C being the shorter isoform generated by use of an alternative translation initiation codon at position 122 . While both isoforms share common C-terminal regions, their distinct N-terminal domains confer different functional properties.

To distinguish between these isoforms experimentally:

• Western blotting using antibodies that recognize both isoforms will show two distinct bands: LAP1B at approximately 68 kDa and LAP1C at approximately 55 kDa

• Isoform-specific antibodies targeting the unique N-terminal region of LAP1B can be used when specific detection is required

• For comprehensive analysis, HPLC-mass spectrometry has proven effective in validating the presence of both isoforms in human cells

• shRNA-mediated knockdown approaches can help verify antibody specificity by demonstrating reduction in both bands

What are the optimal fixation and permeabilization methods when using LAP1 antibodies for immunofluorescence?

For optimal LAP1 antibody performance in immunofluorescence studies, researchers should consider the following methodological approach:

• Fixation: 4% paraformaldehyde for 15-20 minutes at room temperature preserves nuclear membrane structure while maintaining LAP1 antigenicity

• Permeabilization: A dual approach is recommended

  • Initial treatment with 0.1% Triton X-100 for 10 minutes provides access to the nuclear membrane

  • For challenging samples, a follow-up permeabilization with 0.5% saponin may improve antibody penetration to the inner nuclear membrane

• Blocking: 5% BSA in PBS for 1 hour at room temperature minimizes non-specific binding

• Primary antibody incubation: Optimal dilutions typically range from 1:500 to 1:1000, with overnight incubation at 4°C yielding best results

This protocol has been successfully employed in studies examining LAP1 expression in melanoma progression and nuclear envelope dynamics .

What control samples should be included when validating LAP1 antibody specificity?

Rigorous validation of LAP1 antibody specificity requires several key controls:

• Positive controls:

  • Cell lines with confirmed high LAP1 expression (e.g., metastatic melanoma cell lines like WM983B or A375M2)

  • Human skin fibroblasts, which express endogenous LAP1 at detectable levels

• Negative controls:

  • LAP1 knockdown samples using siRNA or shRNA approaches

  • Non-specific IgG of the same species as the primary antibody

  • Peptide competition assays using the immunizing peptide

• Cross-validation:

  • Parallel assays with multiple LAP1 antibodies targeting different epitopes

  • Complementary methods such as western blotting and immunofluorescence

  • Mass spectrometry verification of immunoprecipitated proteins

These controls collectively ensure that signals detected are specific to LAP1 rather than resulting from non-specific antibody interactions.

How can LAP1 antibodies be effectively used to investigate nuclear envelope dynamics in cancer cell migration?

Investigating nuclear envelope dynamics in cancer cell migration using LAP1 antibodies requires specialized experimental approaches:

• Live-cell imaging combined with immunofluorescence:

  • Transfect cells with fluorescently tagged nuclear envelope markers (e.g., LAP1-mRuby3)

  • Perform time-lapse microscopy during constrained migration assays

  • Fix cells at different migration stages and stain with LAP1 antibodies (1:1000 dilution)

  • Quantify nuclear envelope blebs and correlate with migration efficiency

• Transwell migration assays with nuclear envelope analysis:

  • Subject cells to two-round transwell migration assays through 3-μm pores

  • After each round, fix a subset of cells and immunostain for LAP1

  • Analyze nuclear envelope morphology and LAP1 distribution

  • Quantify the percentage of cells exhibiting nuclear envelope blebbing

• Correlative light and electron microscopy:

  • Immunolabel LAP1 with gold-conjugated secondary antibodies

  • Use electron microscopy to visualize ultrastructural changes in the nuclear envelope

  • Correlate LAP1 localization with structural changes during constrained migration

This integrated approach has revealed that LAP1C supports nuclear envelope blebbing during constrained migration and invasion by allowing weaker coupling between the nuclear envelope and nuclear lamina .

What is the significance of LAP1 isoform-specific antibodies in studying melanoma progression?

LAP1 isoform-specific antibodies provide crucial tools for understanding the differential roles of LAP1B and LAP1C in melanoma progression:

These approaches have demonstrated that high LAP1 expression at the invasive front is associated with shorter disease-free survival, suggesting LAP1's potential as a prognostic marker in melanoma .

What are the optimal co-immunoprecipitation conditions for studying LAP1 interactome using antibodies?

Studying the LAP1 interactome requires optimized co-immunoprecipitation (co-IP) conditions:

• Cell lysis buffer optimization:

  • For membrane protein preservation: 1% NP-40 or 0.5% CHAPS in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA

  • Include protease inhibitors (PMSF, aprotinin, leupeptin) and phosphatase inhibitors (sodium orthovanadate, sodium fluoride)

  • For nuclear membrane proteins: consider sonication (3 x 10s pulses) to disrupt nuclear membranes

• Antibody selection and coupling:

  • Use LAP1-specific antibodies (e.g., Proteintech #21459-1-AP)

  • Pre-couple antibodies to protein A/G magnetic beads (10 μg antibody per 50 μl bead slurry)

  • Include rabbit IgG as a negative control for non-specific binding

• Immunoprecipitation procedure:

  • Incubate cell lysates with antibody-coupled beads overnight at 4°C with gentle rotation

  • Wash extensively (at least 4 times) with lysis buffer containing reduced detergent (0.1%)

  • Elute proteins using either low pH buffer or SDS sample buffer for downstream analysis

• Validation and analysis:

  • Perform western blotting to confirm LAP1 pull-down efficiency

  • Subject samples to LC-MS/MS analysis for comprehensive interactome identification

  • Filter identified proteins against control IgG samples to select significantly enriched interactions

This approach has successfully identified 118 proteins as relevant LAP1 interactors in control fibroblasts compared to only 15 LAP1-binding partners in LAP1 E482A mutant fibroblasts , demonstrating the methodology's sensitivity in detecting changes in the LAP1 interactome.

How can phospho-specific LAP1 antibodies be utilized to study post-translational regulation?

Phospho-specific LAP1 antibodies provide valuable tools for investigating post-translational regulation mechanisms:

• Identification of phosphorylation sites:

  • LAP1 undergoes post-translational modifications including phosphorylation and methionine oxidation

  • Phospho-specific antibodies targeting known modification sites can monitor regulatory changes under different cellular conditions

  • For sites regulated by PP1 (protein phosphatase 1), antibodies recognizing specific dephosphorylated residues are particularly valuable

• Experimental approaches:

  • Phosphatase treatment controls: Treating samples with lambda phosphatase before immunoblotting to confirm phospho-specificity

  • Kinase inhibitor studies: Using specific inhibitors to identify kinases responsible for LAP1 phosphorylation

  • Temporal dynamics analysis: Monitoring phosphorylation changes during cell cycle progression or differentiation

  • Mutational analysis: Comparing antibody reactivity between wild-type LAP1 and phospho-site mutants

• Applications in disease models:

  • Comparative phosphorylation analysis between normal and cancer cells

  • Monitoring LAP1 phosphorylation states during neuronal maturation, where LAP1B and LAP1C expression increases

  • Investigating altered phosphorylation in disease-associated LAP1 mutations

• Technical considerations:

  • Rapid sample processing to preserve phosphorylation status

  • Inclusion of phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Careful antibody validation using phospho-mimetic and phospho-dead mutants

These approaches enable researchers to understand how phosphorylation regulates LAP1 function, particularly in contexts like nuclear envelope dynamics and protein interactions.

What are common challenges in detecting LAP1 in tissue samples and how can they be overcome?

Researchers may encounter several challenges when detecting LAP1 in tissue samples:

• Nuclear membrane access limitations:

  • Challenge: LAP1 localizes to the inner nuclear membrane, which can be difficult to access with antibodies

  • Solution: Enhanced permeabilization protocols using 0.5% Triton X-100 for 20 minutes, followed by brief treatment with 0.1% SDS to improve nuclear membrane accessibility

• Epitope masking due to protein-protein interactions:

  • Challenge: LAP1's interactions with lamins, chromatin, and other nuclear envelope proteins may mask antibody epitopes

  • Solution: Employ antigen retrieval methods such as heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95°C for 20 minutes or enzymatic retrieval with proteinase K (10 μg/ml) for 10 minutes

• Isoform-specific detection difficulties:

  • Challenge: Distinguishing between LAP1B and LAP1C in tissue samples

  • Solution: Use isoform-specific antibodies or employ detection protocols that can separate isoforms by molecular weight (e.g., western blotting following tissue protein extraction)

• Variable expression levels:

  • Challenge: LAP1 expression varies across tissues and within tumor regions

  • Solution: Optimize antibody dilutions (typically 1:200-1:500 for immunohistochemistry) and employ digital pathology scoring methods to quantify expression intensity from 0 (very low) to 3 (very high)

• Background reduction:

  • Challenge: Non-specific staining, particularly in tissues with high endogenous peroxidase activity

  • Solution: Include additional blocking steps (10% normal serum from the same species as the secondary antibody) and quench endogenous peroxidases with 3% hydrogen peroxide for 10 minutes before antibody incubation

These optimizations have been successfully applied in studies examining LAP1 expression in melanoma progression, where distinctive staining patterns were observed at the tumor body, proximal invasive front, and distal invasive front .

How can researchers distinguish between specific and non-specific signals when using LAP1 antibodies in complex samples?

Distinguishing specific LAP1 signals from non-specific background requires systematic validation:

• Multiple detection methods approach:

  • Confirm LAP1 detection using complementary techniques (immunofluorescence, western blotting, immunohistochemistry)

  • Verify that the detected molecular weights match expected sizes for LAP1B (~68 kDa) and LAP1C (~55 kDa)

  • Compare subcellular localization patterns across methods (LAP1 should primarily localize to the nuclear envelope)

• RNA interference validation:

  • Perform siRNA or shRNA knockdown of LAP1 (using multiple independent sequences)

  • Compare antibody staining between control and knockdown samples

  • A significant reduction in signal in knockdown samples confirms antibody specificity

• Competitive blocking controls:

  • Pre-incubate the LAP1 antibody with excess immunizing peptide

  • Use this pre-absorbed antibody in parallel with the regular antibody

  • Disappearance of signal with the pre-absorbed antibody indicates specificity

• Cross-validation with recombinant constructs:

  • Express tagged versions of LAP1 (e.g., LAP1-mRuby3)

  • Compare antibody staining with the fluorescent tag signal

  • Co-localization confirms antibody specificity for the target protein

• Mass spectrometry verification:

  • Perform immunoprecipitation with the LAP1 antibody

  • Analyze the precipitated proteins by mass spectrometry

  • Confirmation of LAP1 peptides validates antibody specificity

These approaches collectively provide strong evidence for antibody specificity and allow researchers to confidently interpret LAP1 signals in complex biological samples.

How can LAP1 antibodies be used to study the relationship between nuclear envelope mechanics and cancer cell invasion?

LAP1 antibodies enable sophisticated investigations into nuclear envelope mechanics and cancer invasion:

• Correlative immunofluorescence and biophysical measurements:

  • Stain cells with LAP1 antibodies to visualize nuclear envelope structure

  • Combine with atomic force microscopy to measure nuclear stiffness

  • Correlate LAP1 expression patterns with nuclear deformability metrics

  • This approach has revealed that LAP1C expression allows weaker coupling between the nuclear envelope and nuclear lamina, facilitating nuclear envelope blebbing during constrained migration

• Invasion assay methodologies with immunostaining:

  • Perform 3D collagen I matrix invasion assays with cancer cells

  • Fix and immunostain with LAP1 antibodies at various time points during invasion

  • Analyze nuclear morphology changes and LAP1 distribution during matrix navigation

  • These studies have demonstrated that reducing LAP1 expression levels decreases the ability of metastatic melanoma cells to invade 3D collagen matrices

• Microfluidic constriction assays with real-time imaging:

  • Design microfluidic devices with precisely defined constrictions

  • Express fluorescently-tagged LAP1 constructs or perform live-cell immunostaining

  • Capture real-time nuclear deformation during constriction passage

  • Quantify nuclear rupture frequency, transit time, and recovery kinetics

• In vivo invasion model analysis:

  • Use orthotopic melanoma models where cells are injected into the dermis

  • Analyze three defined regions: tumor body, proximal invasive front, and distal invasive front

  • Apply LAP1 immunohistochemistry and digital pathology to score LAP1 intensity

  • These approaches have shown that LAP1 expression is higher at the proximal invasive front compared to the tumor body and at the distal invasive front compared to the proximal invasive front

These methodologies collectively provide comprehensive insights into how LAP1 contributes to nuclear adaptability during cancer cell invasion.

What are promising approaches for developing LAP1-targeted therapies for aggressive melanoma?

LAP1's emerging role in melanoma progression suggests several therapeutic targeting strategies:

• Structure-based inhibitor design approaches:

  • Use LAP1 antibodies to immunoprecipitate native LAP1 for structural studies

  • Develop compounds targeting the interface between LAP1C and the nuclear lamina

  • Focus on disrupting LAP1's ability to weaken nuclear envelope-lamina coupling

  • Screen candidate compounds using nuclear envelope blebbing assays

• Isoform-specific targeting strategies:

  • Develop therapeutics specifically targeting LAP1C, which supports nuclear envelope blebbing and invasion

  • Create antibody-drug conjugates using LAP1C-specific antibodies to deliver cytotoxic payloads to aggressive melanoma cells

  • Design antisense oligonucleotides or siRNAs targeting LAP1C-specific sequences

• Combination therapy development:

  • Pair LAP1-targeting agents with cytoskeletal inhibitors to comprehensively target cell migration machinery

  • Use LAP1 antibodies in functional assays to identify synergistic drug combinations

  • Screen for compounds that restore normal nuclear envelope-lamina interactions

• Patient stratification methodologies:

  • Develop immunohistochemistry protocols using LAP1 antibodies for diagnostic purposes

  • Create scoring systems based on LAP1 expression at invasive fronts

  • Identify patient subgroups likely to benefit from LAP1-targeted therapies based on expression profiles

These approaches build on the finding that high LAP1 expression is associated with shorter disease-free survival in melanoma patients , suggesting LAP1's potential as both a prognostic marker and therapeutic target.

What experimental conditions should be optimized when using LAP1 antibodies for quantitative analysis of expression levels?

For accurate quantitative analysis of LAP1 expression using antibodies, researchers should optimize several experimental conditions:

• Sample preparation standardization:

  • Cell lysis buffer composition: For western blotting, use LDS buffer 1× with denaturation at 95°C for 5 minutes followed by sonication

  • Protein quantification: Employ BCA or Bradford assays with BSA standards to ensure equal loading

  • Standardized cell densities: Seed cells at consistent densities (e.g., 6 × 10^5 cells/ml in 12-well plates)

• Antibody dilution optimization:

  • Perform titration experiments to determine the linear detection range

  • For western blotting: 1:1,000 dilution of LAP1 antibody (Proteintech #21459-1-AP) typically yields optimal results

  • For immunofluorescence: 1:500 to 1:1,000 dilutions with overnight incubation at 4°C

  • Include appropriate loading controls (GAPDH 1:10,000, MAB374)

• Quantification methodology standardization:

  • For western blotting: Use fluorescent secondary antibodies (IRDye 680RD goat anti-rabbit IgG, 1:10,000) and quantify using systems like Odyssey Fc (LI-COR)

  • For immunohistochemistry: Implement digital pathology scoring systems for consistent intensity evaluation (0-3 scale)

  • For immunofluorescence: Employ automated image analysis with standardized exposure settings

• Calibration and normalization:

  • Include recombinant LAP1 protein standards for absolute quantification

  • Normalize LAP1 expression to appropriate housekeeping controls

  • Consider cell type-specific variations in expression when comparing across samples

• Statistical analysis considerations:

  • Perform multiple independent experiments (minimum n=3)

  • Use appropriate statistical tests based on data distribution

  • Report variability measures (standard deviation or standard error)

These optimizations enable reliable quantitative assessment of LAP1 expression levels across different experimental conditions and cell types.

How can CRISPR-Cas9 genome editing be combined with LAP1 antibodies to study LAP1 function?

CRISPR-Cas9 genome editing provides powerful approaches for studying LAP1 function when combined with antibody-based detection:

• Comprehensive knockout validation strategies:

  • Design guide RNAs targeting different exons of the TOR1AIP1 gene

  • Confirm knockout efficiency using LAP1 antibodies in western blotting

  • Verify loss of both LAP1B and LAP1C isoforms or specific targeting of individual isoforms

  • Assess phenotypic consequences on nuclear envelope structure and cell migration

• Domain-specific functional analysis:

  • Create precise deletions of functional domains (e.g., lamin-binding domain or chromatin-binding region)

  • Generate knock-in cell lines expressing mutations that disrupt specific interactions

  • Compare these engineered lines with cells expressing constructs like LAP1B Δ1-72 or LAP1B ΔCBR

  • Use LAP1 antibodies to confirm expression and proper localization of modified proteins

• Endogenous tagging approaches:

  • Knock in fluorescent tags or epitope tags at the endogenous TOR1AIP1 locus

  • Verify tag incorporation using both tag-specific antibodies and LAP1 antibodies

  • Perform live-cell imaging studies of endogenously tagged LAP1

  • Compare localization and dynamics with antibody staining of fixed cells to validate findings

• Isoform-specific targeting strategies:

  • Design guide RNAs to selectively target LAP1B or LAP1C

  • For LAP1B-specific targeting, design guides targeting the unique N-terminal region

  • For LAP1C-specific targeting, modify the alternative translation initiation site at position 122

  • Validate isoform-specific knockout using antibodies that can distinguish between isoforms

• Rescue experiments with functional readouts:

  • In LAP1 knockout cells, reintroduce wild-type or mutant LAP1 constructs

  • Assess rescue of nuclear envelope morphology, constrained migration, and invasion capacities

  • Use LAP1 antibodies to confirm expression levels comparable to endogenous protein

  • Compare phenotypes with those observed in prior studies of LAP1 function

These approaches leverage the precision of CRISPR-Cas9 editing with the detection capabilities of LAP1 antibodies to gain deeper insights into LAP1 biology.