LMNB1 Antibody, FITC conjugated

What is the biological function of Lamin B1 in nuclear architecture?

Lamin B1 is an integral structural component of the nuclear lamina, a meshwork of proteins located on the nucleoplasmic side of the inner nuclear membrane. Research demonstrates that LMNB1 plays crucial roles in:

• Providing a framework for the nuclear envelope

• Bridging the nuclear envelope and chromatin

• Supporting nuclear assembly and disassembly during cell division

• Regulating chromatin organization and gene expression

• Maintaining telomere dynamics

Studies have shown that LMNB1 is required for proper chromosome condensation in interphase nuclei, and its deficiency can trigger the relocation of epigenetic marks such as H3K27me3 (facultative heterochromatin) toward the interior of the nucleus . Additionally, LMNB1 repression has been observed to slow cellular growth due to S-phase delays and increased genomic instability .

How does FITC conjugation affect the functionality of LMNB1 antibodies?

FITC (Fluorescein isothiocyanate) conjugation provides direct fluorescent labeling of LMNB1 antibodies with the following characteristics:

• Excitation/emission maxima wavelengths of approximately 493-499 nm / 515-522 nm

• Compatible with 488 nm laser lines in flow cytometry and confocal microscopy

• Eliminates the need for secondary antibody incubation steps

When using FITC-conjugated LMNB1 antibodies, researchers should be aware that:

• The conjugation process may slightly alter antibody binding kinetics compared to unconjugated versions

• FITC is sensitive to photobleaching and requires appropriate storage (avoid light exposure)

• FITC's quantum yield and brightness are moderate compared to newer fluorophores

• The pH sensitivity of FITC (optimal at pH 8.0) may affect signal intensity in certain buffer conditions

What distinguishes monoclonal from polyclonal LMNB1 antibodies for research applications?

What are the optimal sample preparation protocols for LMNB1 detection using FITC-conjugated antibodies in immunofluorescence?

For optimal detection of LMNB1 using FITC-conjugated antibodies in immunofluorescence:

Cell Fixation and Permeabilization:

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 in PBS for 10 minutes

• Block with 10% normal serum (goat or donkey) for 30-60 minutes

Antibody Incubation Parameters:

• Primary antibody dilution: 1:50-1:500 (sample-dependent)

• Incubation time: Overnight at 4°C for optimal signal-to-noise ratio

• Buffer composition: PBS with 1-3% BSA and 0.05% Tween-20

Nuclear Counterstaining:

• DAPI (4',6-Diamidino-2-Phenylindole) at 300 nM for 5 minutes before mounting

• Mount with anti-fade reagent (e.g., ProLong Gold) to reduce photobleaching

Imaging Considerations:

• Capture images within the linear fluorescence range

• Use integrated morphometry analysis for quantitative measurements

• Employ appropriate negative controls (isotype control antibody)

How can LMNB1 antibodies be used to investigate changes in nuclear envelope structure during cellular processes?

LMNB1 antibodies are valuable tools for studying nuclear envelope dynamics:

Cell Cycle Analysis:

• Track changes in LMNB1 distribution during mitosis when the nuclear lamina disassembles

• Combine with cell cycle markers (e.g., cyclin antibodies) to correlate LMNB1 patterns with specific phases

• Measure phosphorylation-dependent changes in LAMNB1 organization

Cellular Differentiation Studies:

• Monitor LMNB1 expression and localization changes during stem cell differentiation

• Correlate alterations in nuclear morphology with lineage commitment

• Compare LMNB1 patterns between proliferating and post-mitotic cells

Disease Models:

• Examine LMNB1 organization in models of laminopathies and nuclear envelope disorders

• Study LMNB1 in cancer cells with altered nuclear morphology

• Investigate LMNB1 in models of autosomal dominant leukodystrophy (ADLD)

Methodological Approach:

• Use live-cell imaging with stable cell lines expressing fluorescent-tagged LMNB1

• Apply super-resolution microscopy techniques for detailed lamina structure analysis

• Combine with chromatin immunoprecipitation (ChIP) to map LMNB1-chromatin interactions

• Implement quantitative image analysis for nuclear morphometric measurements

What experimental controls are essential when using FITC-conjugated LMNB1 antibodies in flow cytometry?

For rigorous flow cytometry experiments with FITC-conjugated LMNB1 antibodies:

Essential Controls:

• Unstained Cells: To establish autofluorescence baseline

• Isotype Control: Matched isotype (IgG or IgG1) conjugated to FITC at the same concentration

• Single-Color Controls: When using multiple fluorophores for compensation

• LMNB1 Knockout/Knockdown Cells: Ideal negative control to confirm specificity

• Fixation/Permeabilization Controls: To assess the effect of these treatments on cellular autofluorescence

Optimization Parameters:

• Antibody concentration: 0.40 μg per 10^6 cells in 100 μl suspension

• Fixation: 4% paraformaldehyde followed by permeabilization with permeabilization buffer

• Blocking: 10% normal goat serum to reduce non-specific binding

Gating Strategy:

• Exclude debris using FSC/SSC

• Select single cells using pulse width or height vs. area

• Define positive populations using the controls listed above

• Consider cell cycle phase when analyzing nuclear proteins like LMNB1

Flow cytometry validation data has shown specific detection of LMNB1 in A431 cells with clear separation between stained populations and controls .

How should researchers validate the specificity of LMNB1 antibodies in their experimental systems?

A comprehensive validation strategy for LMNB1 antibodies should include:

Genetic Validation:

• Test in LMNB1 knockout cell lines (such as HAP1 or HeLa LMNB1 KO cells)

• Compare with LMNB1 knockdown cells using targeted siRNA or shRNA

• Perform rescue experiments by re-expressing LMNB1 in knockout cells

Biochemical Validation:

• Western blot analysis to confirm the expected molecular weight (66-72 kDa)

• Immunoprecipitation followed by mass spectrometry identification

• Peptide competition assays with the immunizing peptide

Cross-Reactivity Assessment:

• Test reactivity across multiple species if working with non-human models

• Evaluate potential cross-reactivity with other lamin family members (Lamin A/C, Lamin B2)

• Perform immunostaining in tissues with known LMNB1 expression patterns

Application-Specific Validation:

• For immunofluorescence: co-localization with other nuclear envelope markers

• For ChIP applications: validation of binding to known LMNB1-associated genomic regions

• For flow cytometry: comparison with established nuclear envelope markers

What approaches can be used to study the role of LMNB1 in gene regulation and chromatin organization?

To investigate LMNB1's role in gene regulation and chromatin organization:

ChIP-Seq Analysis:

• Perform chromatin immunoprecipitation with LMNB1 antibodies followed by sequencing

• Cross-link cells with 1% paraformaldehyde for 5 minutes at room temperature

• Sonicate chromatin to ~300 bp fragments using Bioruptor or similar device

• Immunoprecipitate using LMNB1 antibodies immobilized on protein G beads

• Analyze lamina-associated domains (LADs) and compare across conditions

Genome-Nuclear Lamina Interactions:

• Implement DamID (DNA adenine methyltransferase identification) with LMNB1 fusion proteins

• Use Hi-C approaches to map chromatin interactions at the nuclear periphery

• Apply FISH techniques to visualize specific loci relative to the nuclear lamina

Transcriptional Impact Assessment:

• Conduct RNA-Seq following LMNB1 depletion or overexpression

• Analyze changes in expression of genes located in LADs versus non-LADs

• Investigate alternative splicing events affected by LMNB1 levels

Epigenetic Profiling:

• Map distribution of heterochromatin marks (H3K9me3, H3K27me3) relative to LMNB1

• Examine changes in histone modifications following LMNB1 disruption

• Correlate DNA methylation patterns with LMNB1-chromatin associations

Research has demonstrated that LMNB1 knockdown expands CD34+ HSPCs in liquid culture and affects expression of hematopoietic stem cell-specific genes in a dose-dependent manner .

What is the significance of LMNB1 in hematopoietic stem cells and how can researchers best study it?

LMNB1 plays important roles in hematopoietic stem cells (HSCs) with significant research implications:

Key Research Findings:

• LMNB1 knockdown in CD34+ HSPCs enhances self-renewal capacity

• LMNB1-depleted HSPCs gain secondary serial replating potential indicative of increased stemness

• LMNB1 regulates HSC-specific gene signatures in a dose-dependent manner

• LMNB1 deletion has been associated with myeloid neoplasms and nuclear anomalies

• LMNB1 is involved in somatic mutations and progression of B-cell malignancies

Experimental Approaches for LMNB1 Study in HSCs:

• Gene Modulation:

  • Lentiviral shRNA knockdown (achieve different levels: ~75% for low expression, ~50% for intermediate)

  • CRISPR/Cas9 gene editing for complete knockout

  • Inducible systems for temporal control of expression

• Functional Assays:

  • Methylcellulose colony-forming assays to assess clonogenic potential

  • Serial replating to evaluate self-renewal capacity

  • Liquid culture expansion of CD34+ populations

  • Transplantation assays in immunodeficient mice

• Molecular Analysis:

  • RNA-seq to identify gene expression changes

  • ATAC-seq to assess chromatin accessibility alterations

  • ChIP-seq to map LMNB1-chromatin interactions

  • Nuclear morphology assessment using LMNB1 immunofluorescence

• Clinical Correlation:

  • Analysis of LMNB1 expression in patient samples with myeloid disorders

  • Study of 5q deletions (containing LMNB1) in MDS/AML patients

  • Investigation of LMNB1 in patients with complex karyotypes including TP53 mutations

What are common pitfalls when using FITC-conjugated LMNB1 antibodies and how can they be overcome?

Common challenges and their solutions when working with FITC-conjugated LMNB1 antibodies:

Additional troubleshooting approaches:

• For weak nuclear envelope staining, enzyme antigen retrieval with reagents like AR0022 for 15 minutes can enhance signal

• When performing flow cytometry, ensure cells are properly fixed with 4% paraformaldehyde and thoroughly permeabilized

• For multiple channels, carefully design experiments to avoid spectral overlap with FITC (avoid PE or other yellow-green fluorophores)

How can researchers quantitatively analyze LMNB1 distribution and expression levels using imaging techniques?

Quantitative analysis of LMNB1 using imaging techniques:

Image Acquisition Parameters:

• Capture images within the linear fluorescence range to ensure quantitative measurements

• Use consistent exposure settings across all experimental conditions

• Include fluorescence intensity calibration standards

• Acquire z-stacks to capture the full 3D nuclear envelope structure

Quantification Methods:

• Nuclear Rim Measurement: Measure fluorescence intensity around the nuclear periphery

• Rim-to-Nucleoplasm Ratio: Calculate the ratio between nuclear envelope and nucleoplasmic signal

• Integrated Density: Measure total LMNB1 signal per nucleus using integrated morphometry analysis

• Nuclear Volume and Morphology: Assess nuclear size, shape, and LMNB1 distribution simultaneously

Software and Analysis Tools:

• MetaMorph with integrated morphometry analysis module

• ImageJ/FIJI with nuclear analysis plugins

• CellProfiler for high-throughput quantitative image analysis

• MATLAB-based custom analysis for advanced quantification

Statistical Approaches:

• Analyze at least 50-100 cells per condition for robust statistics

• Apply appropriate statistical tests based on data distribution

• Use hierarchical analysis if cells are from different experiments/donors

• Consider machine learning approaches for complex pattern recognition

How should researchers interpret changes in LMNB1 localization or expression in disease models?

When interpreting LMNB1 alterations in disease models, consider:

Cancer and Proliferative Disorders:

• LMNB1 levels often correlate with cell proliferation status

• Changes in LMNB1 distribution may indicate altered nuclear envelope integrity

• LMNB1 loss in certain B-cell malignancies can affect somatic hypermutation

• Decreased LMNB1 may contribute to genomic instability and cancer progression

Hematological Disorders:

• LMNB1 deletion in chromosome 5q is associated with high-risk MDS/AML with TP53 mutations

• Changes in LMNB1 expression can alter hematopoietic stem cell self-renewal and differentiation

• Nuclear shape abnormalities in blood disorders may correlate with LMNB1 alterations

Neurodegenerative Diseases:

• LMNB1 gene duplication causes autosomal dominant leukodystrophy (ADLD)

• Changes in nuclear lamina composition may affect neuronal function and survival

• LMNB1 abnormalities can disrupt neuronal nuclear-cytoskeletal connections

Interpretative Framework:

• Establish normal LMNB1 patterns in relevant cell types/tissues

• Distinguish between primary LMNB1 alterations and secondary effects

• Correlate changes with functional outcomes (proliferation, differentiation, apoptosis)

• Consider compensatory mechanisms involving other lamins (A/C, B2)

• Validate findings across multiple experimental systems and patient samples

How does LMNB1 interact with chromatin remodeling complexes to regulate gene expression?

LMNB1 influences gene expression through various interactions with chromatin:

Mechanistic Interactions:

• LMNB1 anchors heterochromatin to the nuclear periphery through interactions with chromatin-binding proteins

• Lamina-Associated Domains (LADs) are genomic regions that interact with the nuclear lamina and typically show transcriptional repression

• LMNB1 affects distribution of epigenetic marks such as H3K27me3 (facultative heterochromatin)

• Loss of LMNB1 can lead to redistribution of heterochromatin away from the nuclear periphery

Experimental Approaches to Study These Interactions:

• Proximity Labeling Techniques:

  • BioID or APEX2 fused to LMNB1 to identify proximal interacting proteins

  • Analysis of biotinylated proteins by mass spectrometry

• Chromatin Conformation Capture:

  • Hi-C or 4C to map LMNB1-associated chromatin interactions

  • DamID-seq to identify genomic regions contacting the nuclear lamina

• Epigenetic Profiling:

  • ChIP-seq for histone modifications in control vs. LMNB1-depleted cells

  • ATAC-seq to assess changes in chromatin accessibility

  • CUT&RUN for high-resolution mapping of protein-DNA interactions

• Live Cell Dynamics:

  • FRAP (Fluorescence Recovery After Photobleaching) to study LMNB1 mobility

  • Single-molecule tracking to analyze LMNB1-chromatin interactions in real time

What role does LMNB1 play in cellular responses to DNA damage and genomic instability?

LMNB1's involvement in DNA damage response and genomic stability:

Current Research Findings:

• LMNB1 repression leads to S-phase delays and increased genomic instability

• LMNB1 regulates somatic mutations and progression of B-cell malignancies

• Nuclear lamina disruption affects DNA repair pathway choice and efficiency

• LMNB1 may influence telomere maintenance and chromosome end protection

Experimental Approaches:

• DNA Damage Induction and Tracking:

  • UV, ionizing radiation, or chemical damage inducers followed by LMNB1 immunostaining

  • Time-course analysis of LMNB1 redistribution after DNA damage

  • Co-localization with DNA damage markers (γH2AX, 53BP1, RAD51)

• Repair Pathway Analysis:

  • Assess homologous recombination vs. non-homologous end joining in LMNB1-depleted cells

  • Reporter assays for specific repair pathways in LMNB1 mutant cells

  • Analyze recruitment kinetics of repair factors in cells with altered LMNB1

• Genomic Instability Measurement:

  • Micronuclei formation assays in LMNB1-deficient cells

  • Chromosome segregation analysis during mitosis

  • Telomere dysfunction-induced foci (TIF) analysis

• Mechanistic Studies:

  • Investigate post-translational modifications of LMNB1 following DNA damage

  • Assess changes in nuclear mechanics after damage in relation to LMNB1

  • Study chromatin mobility at damage sites in relation to the nuclear lamina

How can advanced imaging technologies be combined with LMNB1 antibodies to reveal novel aspects of nuclear architecture?

Cutting-edge imaging approaches for LMNB1 research:

Super-Resolution Microscopy:

• Structured Illumination Microscopy (SIM): Achieves ~100 nm resolution to resolve fine nuclear lamina structure

• Stochastic Optical Reconstruction Microscopy (STORM): Provides ~20 nm resolution for single-molecule localization of LMNB1

• Stimulated Emission Depletion (STED): Allows visualization of LMNB1 distribution at ~40-50 nm resolution

• Expansion Microscopy: Physical expansion of samples for enhanced resolution of nuclear envelope structures

Correlative Light and Electron Microscopy (CLEM):

• Combine fluorescence imaging of FITC-LMNB1 with electron microscopy ultrastructure

• Use nanogold-conjugated secondary antibodies for precise localization

• Implement cryo-CLEM to preserve native structures

Live Cell Advanced Imaging:

• FLIM (Fluorescence Lifetime Imaging Microscopy) to study LMNB1 protein interactions

• Single-particle tracking of LMNB1 dynamics during cell cycle progression

• Lattice light-sheet microscopy for 3D visualization of nuclear lamina with minimal phototoxicity

Multi-Modal Approaches:

• Combine LMNB1 imaging with mechanical measurements (AFM, micropipette aspiration)

• Implement microfluidic devices to study nuclear deformation and LMNB1 response

• Use optogenetic tools for targeted disruption of nuclear lamina regions

Implementation Strategies:

• Optimize fixation protocols specifically for super-resolution imaging

• Consider dual-color approaches with other nuclear envelope components

• Develop computational analysis pipelines for quantitative assessment of nanoscale LMNB1 distribution

• Use machine learning for pattern recognition in complex nuclear envelope architectures