LDB1 Antibody

What is LDB1 and what are its primary functions in cellular processes?

LDB1, also known as Carboxyl-terminal LIM domain-binding protein 2 (CLIM2), is a nuclear cofactor that interacts with LIM homeodomain proteins to form multiprotein complexes. It regulates transcriptional activity by determining specific partner interactions across multiple developmental pathways .

Key functions include:

• Development of interneurons and motor neurons in cooperation with LHX3 and ISL1

• Axis formation and gene expression activation with LHX1/LIM1

• Regulation of red blood cell development with LMO2, maintaining erythroid precursors in an immature state

• Essential role in the maintenance of both fetal and adult hematopoietic stem cells (HSCs)

• Transcriptional control of AID in B cell class switch recombination

LDB1 is widely expressed in brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, lung, and peripheral blood leukocytes .

What applications are validated for LDB1 antibodies?

LDB1 antibodies are validated for multiple experimental applications:

Recommended positive controls include 293T, A431, HeLa, HepG2 cell lines, and mouse brain tissue .

What species reactivity is reported for LDB1 antibodies?

Commercial LDB1 antibodies demonstrate cross-reactivity with multiple species due to high sequence conservation:

This broad cross-reactivity makes these antibodies valuable for comparative studies across model organisms .

What is the molecular composition and structure of LDB1?

LDB1 is detected at approximately 49 kDa in Western blot applications . The protein contains:

• LIM interaction domain that binds to LIM domains of transcription factors

• Dimerization domain required for homodimerization and chromatin looping functions

• Nuclear localization signals for proper subcellular targeting

Due to alternative splicing events, three isoforms exist for LDB1, with isoform 1 being the most commonly studied . The protein sequence includes the domain: MLDRDVGPTPMYPPTYLEP GIGRHTPYGNQTDYRIFELNKRLQNWTEECDNLWWDAFTTEFFEDDAMLTI TFCLEDGPKRYTIGRTLIP RYFRSIFEGGATELYYVLKH PKEAFHSNFVSLDCDQGSMV TQHGKPMFTQVCVEGRLYLE FMFDDMMRIKTWHFSIRQHR ELIPRSILAMHAQDPQMLDQ LSKNITRCGLSNSTLNYLRLCVI (1-375 aa encoded by BC000482) .

How can LDB1 antibodies be optimized for chromatin immunoprecipitation (ChIP) studies?

Optimizing LDB1 antibodies for ChIP studies requires careful attention to several parameters:

Protocol Recommendations:

• Cross-linking: Use 1% formaldehyde for 10-15 minutes at room temperature

• Chromatin shearing: Optimize sonication to generate 200-500 bp fragments

• Antibody selection: Use ChIP-grade antibodies with validated native protein recognition

• Blocking and washing: Include BSA (typically 3%) in blocking solutions to reduce background

• Controls: Include IgG control and positive control regions (known LDB1 binding sites)

Genome-wide profiling by ChIP-Seq has successfully identified LDB1 complex-binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance . Such studies revealed LDB1 binding at conserved sites in or near 20 of 28 HSC genes analyzed and in 11 of 12 known enhancer elements in proximity to these genes .

What is known about the role of LDB1 in regulating chromatin architecture?

LDB1 plays crucial roles in chromatin architecture and gene regulation:

• Long-range DNA interactions: LDB1 is required for the relocalization of the β-globin locus to the nuclear interior and establishes spatial proximity between the Locus Control Region (LCR) and β-globin gene .

• Chromatin looping: Through its dimerization domain, LDB1 mediates loops between regulatory elements, as observed in:

  • The β-globin locus during erythroid development

  • The AID promoter and upstream enhancers in B cells

• Transcription factor complex stabilization: In studies of erythroid cells, knockdown of Ldb1 reduced occupancy of the entire complex (LMO2, SCL, and GATA-1) at regulatory sites between 2- and 7-fold, indicating that LDB1 stabilizes the entire complex on chromatin .

Research demonstrates that shRNA-mediated reduction of Ldb1 prevents interaction between the LCR and β-globin promoter, highlighting its role as an architectural protein .

How does LDB1 function differ between hematopoietic lineages?

LDB1 exhibits distinct functions across hematopoietic lineages:

Unlike in other hematopoietic lineages where LDB1 and LMO2 function together, in B cells they operate separately, with LDB1 regulating transcription and LMO2 contributing to DNA repair mechanisms .

What methods can validate LDB1 antibody specificity in experimental systems?

To ensure LDB1 antibody specificity:

Validation approaches:

• Genetic validation:

  • Test antibodies in Ldb1 knockout models, such as the conditional mutants described using the En1cre system

  • Compare staining in control versus Ldb1 knockdown cells established using lentiviral shRNAs

• Biochemical validation:

  • Western blot should show a single band at 49 kDa

  • Immunoprecipitation followed by mass spectrometry to confirm pulled-down protein identity

  • Peptide competition assays with the immunizing peptide

• Cross-platform validation:

  • Compare results across multiple techniques (IF, WB, IP, IHC)

  • Test multiple antibodies targeting different epitopes

  • Include positive controls (293T, A431, HeLa, HepG2, Mouse brain)

For immunofluorescence validation specifically, researchers have successfully used a protocol with 4% PFA fixation, 1% SDS antigen retrieval, and 0.3% Triton X-100 with 3% BSA blocking .

How do LDB1 and LMO2 interact in multiprotein complexes?

In most hematopoietic lineages, LDB1 and LMO2 form a core complex with additional transcription factors:

• Complex composition:

  • LDB1 (nuclear adaptor)

  • LMO2 (non-DNA-binding adaptor)

  • E2A (transcription factor)

  • Scl/TAL1 (transcription factor)

  • GATA-1 or GATA-2 (transcription factors)

• Protein stability regulation:

  • LDB1 stabilizes LMO2 proteins in most contexts

  • LDB1 knockdown reduces occupancy of LMO2, SCL, and GATA-1 at chromatin, indicating its role in stabilizing the entire complex

• Functional independence in B cells:

  • Unlike in other hematopoietic lineages, in B cells, LDB1 and LMO2 function separately

  • LMO2 contributes to class switch recombination by promoting end joining of DNA double-strand breaks

  • LDB1 regulates AID transcription independently of LMO2

This functional relationship can be studied using co-immunoprecipitation with LDB1 antibodies to pull down associated proteins, followed by Western blotting for complex members.

What role does LDB1 play in DNA repair and class switch recombination?

Recent research has revealed distinct roles for LDB1 in B cell class switch recombination (CSR):

• Mechanistic separation from LMO2:

  • LMO2 contributes to CSR by promoting end joining of DNA double-strand breaks (DSBs) and inhibiting end resection

  • LDB1, while stabilizing LMO2 proteins, is not required for end joining

• Transcriptional regulation:

  • LDB1 functions as a positive regulator of AID transcription independent of LMO2

  • This function requires LDB1's dimerization domain

  • LDB1 directly binds to and promotes the looping of the AID promoter to upstream enhancers through dimerization

• CRISPR/Cas9 screening validation:

  • Both LMO2 and LDB1 were identified as factors for class switch recombination through CRISPR/Cas9-based loss-of-function screening in murine B cells

These findings highlight the mechanistically separated roles of LDB1 and LMO2 in different steps of CSR for antibody diversification.

What structural features of LDB1 are essential for its various functions?

Several domains and structural features are critical for LDB1's diverse functions:

• Dimerization domain:

  • Essential for chromatin looping activities

  • Required for regulation of AID transcription in B cells

  • Mediates homodimerization with LMX1A through strong interaction with the LIM2 domain

• LIM interaction domains:

  • Bind to LIM domains of transcription factors

  • Determine specific partner interactions in different cellular contexts

  • Important for complex formation with LMO2 and other factors in hematopoietic lineages

• Nuclear localization signals:

  • Ensure proper subcellular targeting

  • Critical for transcriptional regulatory functions

Structure-function studies can employ domain-specific antibodies or domain deletion/mutation constructs to determine which regions are necessary for specific interactions or functions.

How can LDB1 antibodies be utilized in studying neurodevelopmental processes?

LDB1 plays important roles in neural development, particularly through its interactions with LIM homeodomain proteins:

• Neuronal development applications:

  • Study interneuron and motor neuron development with LDB1 antibodies

  • Investigate cooperation with LHX3 and ISL1 in neuronal specification

• Recommended methodologies:

  • Immunofluorescence protocol:

    • Fix cells with 4% PFA in PBS (pH 7.4) for 20 min at room temperature

    • Use 1% sodium dodecyl sulfate for 5 min for antigen retrieval

    • Block with 0.3% Triton X-100 and 3% BSA for 45 min

    • Incubate with primary LDB1 antibody overnight at 4°C

    • Counterstain with DAPI after secondary antibody incubation

• Genetic approaches:

  • Conditional knockout models such as En1cre/Ldb1f/f to study isthmic organizer and midbrain/hindbrain development

  • Cell-specific Cre lines to target LDB1 deletion in specific neuronal populations

LDB1 antibodies can reveal changes in expression patterns during neural development and help identify cells where LDB1-dependent transcriptional regulation is active.