LMNB1 Antibody

What is the expected molecular weight of LMNB1 and what control samples should be used for antibody validation?

LMNB1 is a nuclear envelope protein with a canonical length of 586 amino acid residues and an expected molecular weight of 66.4 kDa, though observed bands typically range from 66-70 kDa on Western blots . For proper validation:

• Positive controls: HeLa cells, U-937 cells, K-562 cells, Y79 cells, Jurkat cells, and MCF-7 cells consistently show strong LMNB1 expression

• Tissue controls: Mouse spleen, pancreas, kidney, lung, and eye tissues are recommended positive controls

• Knockout validation: When possible, LMNB1 knockout cell lines provide the strongest validation for antibody specificity

• Cross-reactivity testing: Validated LMNB1 antibodies should be tested across multiple species if cross-species applications are intended

Most commercially available LMNB1 antibodies show reactivity with human, mouse, and rat samples, with some extending to monkey, canine, porcine, and other mammalian species .

What applications are LMNB1 antibodies suitable for and what are the optimal sample preparation methods?

LMNB1 antibodies are versatile tools applicable across multiple experimental platforms:

For IHC applications, colon adenocarcinoma, breast, fallopian tube, tonsil, and testis tissues serve as effective positive controls .

How should LMNB1 antibodies be used to study changes in nuclear lamina organization during cellular senescence?

LMNB1 exhibits dynamic changes during cellular senescence that can be effectively visualized and quantified:

• Comparative Analysis Approach:

  • Create paired samples of growing cells and senescent cells (induced by oncogenic stress, replicative exhaustion, or other triggers)

  • Process both samples identically for either IF, IHC, or Western blot

  • For IF/IHC: quantify nuclear rim intensity and distribution pattern

  • For Western blot: normalize to loading controls and compare relative expression levels

• Single-Cell Analysis Method:

  • Use laser-scanning cytometry (LSC) to correlate LMNB1 levels with senescence markers at the single cell level

  • This method revealed that SAHF-positive nuclei (senescence-associated heterochromatic foci) predominantly appear in LMNB1-low senescent populations

Research has consistently shown that LMNB1 protein levels are reduced during oncogenic H-Ras-induced senescence (RIS) and replicative exhaustion senescence but remain unchanged in quiescent cells induced by serum starvation .

What methodological approaches can address experimental variability when using LMNB1 antibodies in chromatin immunoprecipitation studies?

ChIP experiments with LMNB1 antibodies present unique challenges due to global reduction of LMNB1 levels in certain conditions:

• Normalization Strategies for ChIP-seq:

  • Implement both intercondition and intracondition normalization to account for differences in ChIP-seq library complexity

  • Use spike-in controls (e.g., Drosophila chromatin) for accurate quantitative comparisons

  • Validate ChIP-seq findings with independent ChIP-qPCR experiments for selected genomic regions

• Validation and Controls:

  • Compare LMNB1 ChIP-seq results with DamID (DNA adenine methyltransferase identification) technique using Dam-LMNB1 fusion proteins

  • Expect approximately 80% overlap between DamID-defined LADs (Lamina-Associated Domains) and ChIP-seq results

  • Include regions with stable LMNB1 binding as internal controls

Studies have demonstrated that despite global LMNB1 reduction during senescence, changes in LMNB1 chromatin binding are not uniform across the genome, with preferential reduction from H3K9me3-enriched regions .

How can LMNB1 antibodies be utilized to study the relationship between nuclear lamina alterations and cancer progression?

LMNB1 has significant implications in cancer biology that can be studied through several methodological approaches:

Current research indicates that LMNB1 may function as a "mutational gatekeeper" in B-cell malignancies, suppressing aberrant mutations that drive lymphoid malignancy .

What methodological approaches can resolve contradicting findings about LMNB1 overexpression effects on cellular senescence?

The literature contains apparently contradictory findings regarding LMNB1 overexpression:

• Standardized Experimental Framework:

  • Match cell types, passage numbers, and expression levels across studies

  • Use inducible expression systems to control timing and level of LMNB1 expression

  • Measure multiple senescence markers (β-galactosidase, SAHF, p16, p21)

  • Initial studies showed LMNB1 overexpression increased proliferation and delayed senescence onset, while more recent investigations suggest it can actually induce senescence

• Mechanistic Resolution Approach:

  • Examine dose-dependent effects by creating a gradient of LMNB1 expression levels

  • Analyze interaction with other lamins (Lamin A/C) and nuclear envelope proteins

  • Investigate context-dependent effects in different cell types and under various stress conditions

  • Study the combined effects of LMNB1 with other factors like HMGA proteins to fully understand mechanisms of SAHF formation

• Time-Course Analysis:

  • Implement time-resolved single-cell tracking following LMNB1 manipulation

  • This approach can distinguish between initial, intermediate, and long-term effects of LMNB1 alterations

How can LMNB1 antibodies be employed to study the relationship between nuclear lamina and somatic hypermutation in B-cells?

LMNB1 has been identified as a negative epigenetic regulator of somatic hypermutation (SHM) in B-cells:

What are the optimal approaches for studying LMNB1's role in chromatin organization through LAD dynamics?

Lamina-Associated Domains (LADs) are genomic regions that interact with the nuclear lamina:

• Integrated LAD Mapping Strategy:

  • Combine LMNB1 ChIP-seq with DamID techniques for comprehensive LAD identification

  • Integrate with histone modification mapping (H3K9me3, H3K27me3)

  • Analysis reveals H3K9me3 enrichment in central regions of LADs and H3K27me3 enrichment at LAD edges

• LAD Release Visualization Technique:

  • Implement DNA fluorescence in situ hybridization (FISH) with BAC probes mapped to genomic regions

  • Track physical movement of specific genomic loci during LMNB1 reduction

  • Correlation exists between LMNB1 density associated with DNA regions and their perinuclear localization

• Mechanistic Intervention Protocol:

  • Manipulate LMNB1 levels through RNAi or overexpression

  • Combine with manipulation of chromatin modifiers (like HMGA proteins)

  • Co-expression of sh-LMNB1 and HMGA proteins efficiently induces senescence-associated heterochromatic foci (SAHFs)

What are the critical factors for optimizing LMNB1 antibody performance across different experimental platforms?

Successful LMNB1 antibody application requires platform-specific optimizations:

Additional considerations include antibody concentration optimization, incubation times and temperatures, and blocking reagent selection to minimize background.

How should discrepancies in experimental results using different LMNB1 antibodies be addressed and resolved?

When facing inconsistent results with different LMNB1 antibodies:

• Epitope Mapping Analysis:

  • Compare the immunogen sequences/regions targeted by different antibodies

  • Some antibodies target N-terminal regions while others target C-terminal regions (e.g., ab65986 targets synthetic peptide within Human LMNB1 aa 550 to C-terminus)

  • Post-translational modifications near epitopes may affect antibody recognition

• Validation Hierarchy Implementation:

  • Establish a clear validation hierarchy with knockout controls at the top

  • Secondary validation through siRNA knockdown

  • Tertiary validation through known positive/negative tissue controls

  • Polyclonal antibodies may recognize multiple epitopes while monoclonals target single epitopes

• Multi-antibody Consensus Approach:

  • For critical findings, verify with at least two antibodies recognizing different epitopes

  • Document specific lot numbers, as antibody performance can vary between lots

  • Consider using multiple detection methods (fluorescence, chromogenic, etc.)