SSBP3 Antibody, HRP conjugated

What is SSBP3 and what are its primary biological functions?

SSBP3 (single-stranded DNA binding protein 3) is a critical regulatory protein involved in multiple cellular processes. At the molecular level, SSBP3 functions primarily as:

• An inhibitor of proteasomal degradation of specific transcription factors, particularly Lhx2 and Ldb1

• A promoter of DNA-binding complex assembly

• A regulator of gene transcription through recruitment to specific promoters, such as the Cga promoter

• A component of transcriptional complexes containing LIM-homeodomain proteins

Research has revealed that SSBP3 is highly expressed in excitatory glutamatergic neurons and oligodendrocytes in the human brain, though at relatively lower levels compared to neuronal markers . The protein contains highly conserved domains, including a LisH domain (approximately 97% identical between human and Drosophila) and a proline-rich domain (approximately 54% identical) .

In pancreatic β-cells, SSBP3 interacts with Ldb1 and Isl1 to regulate expression of critical genes, including MafA and Glp1r . These multifaceted roles highlight SSBP3's importance in diverse developmental and physiological contexts.

What are the specifications and applications of SSBP3 Antibody, HRP conjugated?

SSBP3 Antibody, HRP conjugated is a polyclonal antibody with specific reactivity against human SSBP3 samples . The key specifications include:

• Host: Rabbit

• Clonality: Polyclonal

• Isotype: IgG

• Conjugation: Horseradish Peroxidase (HRP)

• Epitope: Internal region of SSBP3

The antibody has been tested and validated for the following applications:

• ELISA: Recommended dilution of 1:1000

• Western Blot: Recommended dilution of 1:100-500

The HRP conjugation enables direct detection without the need for secondary antibodies, streamlining experimental workflows and potentially reducing background issues in applications like ELISA and western blotting.

How can SSBP3 antibody be used to investigate protein-protein interactions in transcriptional complexes?

SSBP3 antibody can be effectively utilized to investigate protein-protein interactions within transcriptional complexes through several methodological approaches:

Co-Immunoprecipitation (Co-IP):

• Lyse cells expressing SSBP3 and potential interacting partners in a non-denaturing buffer to preserve protein-protein interactions

• Incubate cell lysate with SSBP3 antibody coupled to agarose or magnetic beads

• Wash to remove non-specific binding

• Elute bound proteins and analyze by western blotting for suspected interacting partners

As demonstrated in research, this approach has successfully shown that SSBP3 interacts with Ldb1 and Isl1 in β-cell lines and in mouse and human islets . Similarly, interactions between SSBP3, Lhx2, and Ldb1 have been demonstrated in pituitary cells .

Electrophoretic Mobility Shift Assay (EMSA) with Antibody Supershift:

• Prepare nuclear extracts from cells expressing SSBP3

• Incubate with labeled DNA probe containing the binding site of interest

• Add SSBP3 antibody to the reaction

• Analyze by non-denaturing gel electrophoresis

This approach has been effectively used to show that SSBP3 antibody retarded the migration of protein-DNA complexes in αT3-1 pituitary cells, confirming SSBP3's presence in these complexes . Specifically, the complex was retarded by antibody to SSBP3 but not by rabbit IgG or antibody to SSBP2 .

Chromatin Immunoprecipitation (ChIP):

• Cross-link proteins to DNA in intact cells

• Lyse cells and shear chromatin

• Immunoprecipitate with SSBP3 antibody

• Reverse cross-linking and purify DNA

• Analyze by PCR or sequencing

ChIP analysis using SSBP3 antibody has demonstrated that SSBP3 occupies the Cga promoter alongside Lhx2 and Ldb1 in vivo , providing evidence for its role in transcriptional regulation.

What methodologies best assess SSBP3's impact on protein stability and turnover?

Several experimental approaches can effectively measure SSBP3's impact on protein stability and turnover:

Cycloheximide Chase Assay:

• Treat cells with cycloheximide (CHX) to inhibit protein synthesis

• Collect cell lysates at various time points (0, 2, 4, 8 hours)

• Analyze protein levels by western blotting

• Compare protein degradation rates with and without SSBP3 manipulation

This methodology has revealed that SSBP3 knockdown markedly accelerates the turnover of both Ldb1 and Lhx2 protein levels in αT3-1 cells treated with CHX . The quantitative analysis of protein levels over time provides direct evidence of SSBP3's role in preventing protein degradation.

Proteasome Inhibition Assay:

• Treat cells with a proteasome inhibitor (e.g., MG132)

• Compare protein accumulation patterns in control vs. SSBP3-overexpressing cells

• Analyze by western blotting for proteins of interest

Research has shown that MG132 treatment increases the abundance of both Ldb1 and Lhx2 in αT3-1 cells, similar to the effect of enforced SSBP3 expression . This demonstrates that SSBP3 likely functions by inhibiting proteasomal degradation.

Expression Analysis with SSBP3 Manipulation:

• Overexpress or knock down SSBP3 in cell culture

• Measure mRNA and protein levels of suspected targets

• Compare changes at the transcriptional vs. translational level

Studies implementing this approach have shown that while SSBP3 manipulation affects protein levels of targets like Lhx2 and Ldb1, their mRNA levels remain unchanged , supporting a post-transcriptional mechanism of action.

How can SSBP3 antibody be used to investigate neurodevelopmental disorders?

SSBP3 antibody can be employed in several sophisticated experimental approaches to investigate the role of SSBP3 in neurodevelopmental disorders:

Immunohistochemistry in Patient-Derived Samples:

• Obtain brain tissue sections from patients with neurodevelopmental disorders and matched controls

• Perform immunostaining with SSBP3 antibody

• Quantify expression levels and localization patterns

• Correlate with clinical phenotypes and genetic data

This approach could build on findings that SSBP3 is expressed in excitatory neurons in humans , potentially revealing altered expression or localization in pathological conditions.

Functional Studies in Model Systems:

• Create genetic models with altered SSBP3 expression (overexpression or knockdown)

• Assess morphological, physiological, and behavioral phenotypes

• Use SSBP3 antibody to confirm expression changes and identify affected pathways

Research in Drosophila has demonstrated that manipulation of Ssdp (the SSBP3 ortholog) affects brain development, glial cell numbers, and synaptic density . Specifically, Ssdp overexpression caused morphological alterations in Drosophila wing, mechanosensory bristles, and head, while also affecting neuropil brain volume and glial cell number in larvae and adult flies .

Molecular Pathway Analysis:

• Use SSBP3 antibody in ChIP-seq experiments to identify genome-wide binding sites

• Compare binding patterns between control and disease models

• Integrate with transcriptomic data to identify dysregulated pathways

This comprehensive approach could extend observations that SSBP3 influences canonical Wnt signaling, which plays crucial roles in neurodevelopment .

The connection to neurodevelopmental disorders is particularly relevant as the 1p32.3 chromosomal region harboring SSBP3 has been implicated in conditions characterized by developmental delay, intellectual disability, autism, and macro/microcephaly .

What experimental approaches can distinguish between direct and indirect effects of SSBP3 on transcriptional regulation?

Distinguishing direct from indirect effects of SSBP3 on transcriptional regulation requires sophisticated experimental designs:

Sequential ChIP (Re-ChIP):

• Perform initial ChIP with antibodies against SSBP3

• Re-immunoprecipitate with antibodies against suspected partner proteins (e.g., Ldb1, Lhx2)

• Analyze enriched DNA regions by qPCR or sequencing

This approach can determine whether SSBP3 and its interaction partners simultaneously occupy the same genomic regions, supporting direct regulation. Research has already established that SSBP3, Ldb1, and Lhx2 occupy the Cga promoter , and this technique would further clarify their co-occupancy.

Domain-Specific Mutational Analysis:

• Generate SSBP3 constructs with mutations in specific functional domains

• Express these constructs in cells with knocked-down endogenous SSBP3

• Assess protein interactions, DNA binding, and transcriptional activity

• Use SSBP3 antibody to confirm expression of mutant proteins

This approach leverages the understanding that SSBP3 contains highly conserved domains, including the LisH domain and proline-rich domain . Studies have noted the importance of the LUFS domain in mediating interaction with Ldb1 , making it a prime target for mutational analysis.

Temporal Manipulation of SSBP3 Expression:

• Employ inducible expression or knockdown systems to control SSBP3 levels

• Monitor short-term vs. long-term effects on target gene expression

• Compare immediate transcriptional changes with secondary effects

This strategy builds on observations that temporal SSBP3 knockdown in adult Drosophila did not produce behavioral and functional defects, unlike developmental knockdown , suggesting stage-specific roles.

What are common challenges when using SSBP3 antibody in chromatin immunoprecipitation, and how can they be addressed?

Chromatin immunoprecipitation (ChIP) with SSBP3 antibody can present several challenges that require specific optimization strategies:

Challenge: Low Signal-to-Noise Ratio
Solutions:

• Optimize antibody concentration based on titration experiments (typically start with 1-5 μg per ChIP reaction)

• Increase stringency of wash buffers to reduce non-specific binding

• Implement sequential ChIP approaches to increase specificity when studying complex formation

Challenge: Variability in Chromatin Shearing
Solutions:

• Optimize sonication conditions for each cell or tissue type

• Verify fragment sizes (aim for 200-500 bp) by agarose gel electrophoresis

• Implement controlled cross-linking conditions (typically 1% formaldehyde for 10 minutes)

Challenge: Limited Specificity Validation
Solutions:

• Include negative control regions in qPCR analysis (as demonstrated in the research where SSBP3 antibody did not precipitate chromatin fragments from the 3' UTR of Cga)

• Use isotype control antibodies (rabbit IgG) as negative controls

• Validate findings with complementary approaches such as EMSA

Table 1: Recommended ChIP Protocol Modifications for SSBP3 Antibody

These recommendations are based on successful ChIP experiments that demonstrated SSBP3 occupancy on the Cga promoter alongside Lhx2 and Ldb1 .

How can researchers validate SSBP3 antibody specificity for their particular experimental system?

Validating antibody specificity is crucial for research integrity. For SSBP3 antibody, consider these methodological approaches:

Genetic Knockdown/Knockout Controls:

• Generate SSBP3 knockdown using siRNA or shRNA approaches

• Perform western blot or immunostaining with SSBP3 antibody

• Confirm reduction in signal intensity corresponding to knockdown efficiency

This approach builds on methods used in studies where SSBP3 knockdown demonstrated effects on Lhx2 and Ldb1 protein levels .

Peptide Competition Assay:

• Pre-incubate SSBP3 antibody with excess purified SSBP3 peptide (matching the epitope)

• In parallel, prepare identical samples with non-blocked antibody

• Compare signal reduction in the blocked vs. non-blocked conditions

• A specific antibody will show significantly reduced signal when blocked with its target peptide

Orthogonal Detection Methods:

• Express tagged SSBP3 (e.g., FLAG or HA tag)

• Perform parallel detection with both anti-tag antibody and SSBP3 antibody

• Compare localization and expression patterns

• Concordant results support antibody specificity

Western Blot Analysis:

• Run protein samples from multiple cell types with known SSBP3 expression levels

• Probe with SSBP3 antibody at recommended dilutions (1:100-500)

• Verify single band at the expected molecular weight

• Compare band intensity with known expression differences (e.g., higher in neural tissues)

How is SSBP3 involved in pancreatic β-cell function, and what research methodologies best explore this connection?

Recent research has revealed important roles for SSBP3 in pancreatic β-cell function through several key mechanisms:

SSBP3-Ldb1-Isl1 Regulatory Complex:
Studies have confirmed that SSBP3 interacts with Ldb1 and Isl1 in β-cell lines and in mouse and human islets . This interaction appears critical for proper β-cell function through regulation of essential genes.

Target Gene Regulation:
SSBP3 has been shown to occupy promoters of key β-cell genes, including MafA and Glp1r, alongside Ldb1 and Isl1 . Knockdown of SSBP3 in β-cell lines imparts mRNA deficiencies similar to those observed upon Ldb1 reduction .

Methodological Approaches for Further Research:

• Single-Cell Transcriptomics in Pancreatic Islets:

  • Isolate islet cells from control and diabetic models

  • Perform single-cell RNA sequencing

  • Analyze SSBP3 co-expression patterns with β-cell markers

  • Identify cell-type specific regulatory networks

• Conditional Knockout Models:

  • Generate β-cell-specific SSBP3 knockout mice

  • Assess glucose homeostasis, insulin secretion, and β-cell mass

  • Perform transcriptomic analysis to identify altered pathways

  • Use SSBP3 antibody to confirm deletion specificity

• Chromatin Landscape Analysis:

  • Perform ChIP-seq with SSBP3 antibody in β-cells

  • Integrate with ATAC-seq data to identify accessible chromatin regions

  • Map the complete regulatory network involving SSBP3, Ldb1, and Isl1

  • Correlate with expression changes in diabetic conditions

These approaches would extend current understanding that "SSBP3 is a critical component of Ldb1-Isl1 regulatory complexes, required for expression of critical β-cell target genes" .

What do recent studies reveal about SSBP3's role in neurodevelopment and its potential implications for autism spectrum disorders?

Recent studies have provided compelling evidence linking SSBP3 to neurodevelopment and autism spectrum disorders (ASD):

Genetic Association:
The 1p32.3 chromosomal region harboring SSBP3 has been implicated in neurodevelopmental disorders characterized by developmental delay, intellectual disability, autism, and macro/microcephaly . SSBP3 has been identified as one of 321 candidate genes prioritized in a cross-disorder analysis of de novo mutations, showing significant genetic association with neurodevelopmental disorders such as ASD and intellectual disability .

Expression Pattern:
SSBP3 is expressed in excitatory neurons in the human brain, colocalizing with markers of excitatory glutamatergic neurons (SLC17A7, CUX2, and RORB) and with oligodendrocytes . This expression pattern positions SSBP3 to influence neuronal development and function.

Functional Studies in Drosophila:
Research using Drosophila models has demonstrated that manipulation of Ssdp (the SSBP3 ortholog) affects:

• Neuropil brain volume and glial cell number in larvae and adult flies

• Synaptic density in specific brain regions

• Canonical Wnt signaling, potentially through effects on armadillo levels

• Autism-associated behaviors

Key Methodological Insights for Future Research:

• Optogenetic Manipulation:
  Studies have shown that "optogenetic manipulation of Ssdp-expressing neurons altered autism-associated behaviors" , suggesting that this approach can provide valuable insights into SSBP3's role in neural circuits.

• Temporal-Specific Manipulation:
  Interestingly, "knockdown exclusively in adult flies did not produce behavioral and functional defects" , indicating that SSBP3's role in neurodevelopment may be stage-specific. This suggests that developmental time windows should be carefully considered in future studies.

• Glial Focus:
  The observation that Ssdp manipulation affects glial cell numbers points to the importance of examining not just neuronal effects but also glial contributions to neurodevelopmental disorders.

As research continues, these findings suggest that SSBP3 is a promising candidate for further investigation in the context of neurodevelopmental disorders, particularly autism spectrum disorders.