SSBP3 Antibody, FITC conjugated

What is SSBP3 and what are its primary functions in cellular biology?

SSBP3 (Single-stranded DNA-binding protein 3) belongs to a small family of transcriptional coregulators (including SSBP2-SSBP4) that directly bind to the LCCD domain of Ldb1 and mediate LIM factor complex stability and function . The protein was originally identified by homology to SSDP, a chicken ortholog shown to bind pyrimidine-rich elements within promoter regions . SSBP3 plays critical roles in transcriptional regulation, particularly in the regulation of genes such as alpha 2(I) collagen where it binds to single-stranded polypyrimidine sequences in the promoter region . Recent research has demonstrated that SSBP3 interacts with Ldb1 and Isl1 in pancreatic β-cells, suggesting its importance in maintaining β-cell function . Additionally, SSBP3 has been identified as a regulator for mouse embryonic stem cells (ESCs) to differentiate into trophoblast-like cells .

What are the key technical specifications of the SSBP3 Antibody, FITC conjugated?

The SSBP3 Antibody with FITC conjugation has the following specifications:

The antibody is supplied in a buffer containing 0.01 M PBS, pH 7.4, 0.03% Proclin-300, and 50% glycerol .

How should SSBP3 Antibody, FITC conjugated be stored to maintain optimal activity?

For optimal preservation of antibody activity, the SSBP3 Antibody, FITC conjugated should be aliquoted upon receipt and stored at -20°C . Repeated freeze/thaw cycles should be avoided as they can compromise antibody integrity and fluorophore stability . The glycerol in the buffer (50%) helps prevent complete freezing at -20°C, which reduces damage during freeze/thaw cycles. When planning experiments, it's advisable to thaw only the amount needed for immediate use and keep the working aliquot protected from light to prevent photobleaching of the FITC fluorophore.

How does SSBP3 interact with Ldb1 and Isl1 in transcriptional regulation networks?

Research has demonstrated that SSBP3 is a critical component of Ldb1-Isl1 regulatory complexes in pancreatic β-cells . The interaction was confirmed using reversible cross-link immunoprecipitation (ReCLIP) and mass spectrometry in βTC-3 cell lines, as well as in mouse and human islets . This interaction appears to be crucial for the expression of key β-cell target genes.

Mechanistically, SSBP3:

• Co-occupies known Ldb1-Isl1 target promoters, including MafA and Glp1r

• Contributes to large regulatory complexes with Ldb1 and Isl1 (confirmed by sucrose sedimentation data)

• Supports similar gene expression patterns as Ldb1, as knockdown of SSBP3 imparts mRNA deficiencies similar to those observed upon Ldb1 reduction

These findings suggest that SSBP3 functions as a stabilizing factor in transcriptional complexes, potentially enhancing DNA binding specificity or modulating the activity of other transcriptional regulators within the complex.

What role does SSBP3 play in embryonic stem cell differentiation?

SSBP3 has been identified as a regulator for mouse embryonic stem cell (ESC) differentiation into trophoblast-like cells . Forced expression of Ssbp3 in mouse ESCs upregulates expression levels of lineage-associated genes, with trophoblast cell markers showing the highest elevation . Conversely, depletion of Ssbp3 attenuates the expression of trophoblast lineage marker genes that would normally be induced by downregulation of Oct4 or treatment with BMP4 and bFGF in ESCs .

Interestingly, global gene expression profiling analysis indicated that Ssbp3 overexpression does not significantly alter the transcript levels of pluripotency-associated transcription factors . Instead, Ssbp3 promotes the expression of early trophectoderm transcription factors such as Cdx2 and activates MAPK/Erk1/2 and TGF-β pathways . Furthermore, overexpression of Ssbp3:

• Reduces the methylation level of the Elf5 promoter

• Promotes the generation of teratomas with internal hemorrhage, indicative of the presence of trophoblast cells

These findings position SSBP3 as a potential molecular switch in determining cell fate decisions during early embryonic development.

What is the optimal protocol for ChIP experiments using SSBP3 Antibody?

Based on published research methodologies, the following protocol has been successfully used for chromatin immunoprecipitation (ChIP) experiments with SSBP3 antibody:

• Culture cells to appropriate density (approximately 2 × 10^6 cells per 10-cm dish)

• Cross-link with 1% formaldehyde in DMEM at room temperature

• Prepare protein-DNA chromatin fragments by sonication (e.g., using Bioruptor XL)

• Preclear chromatin with Protein G Dynabeads for 2 hours at 4°C

• Incubate precleared chromatin overnight at 4°C with:

  • α-SSBP3 rabbit polyclonal antibody (controls should include normal rabbit IgG or no antibody)

• Precipitate antibody-bound chromatin complexes with Protein G Dynabeads at 4°C for 4 hours

• Wash complexes, elute from beads, and reverse cross-links

• Perform qPCR on the immunoprecipitated DNA using appropriate primers

When analyzing SSBP3 occupancy, researchers have successfully used primers for genes like MafA Region 3 and Glp1r . For proper controls, regions not expected to bind SSBP3 (like distal regions of the albumin gene) have been utilized.

What are the optimal dilutions and controls for immunofluorescence experiments with SSBP3 Antibody, FITC conjugated?

While the optimal dilutions should be determined by the end user for each specific application , general guidelines for immunofluorescence experiments with SSBP3 Antibody, FITC conjugated include:

Starting dilution recommendations:

• For immunocytochemistry: 1:50 to 1:200

• For flow cytometry: 1:100 to 1:500

Essential controls to include:

• Isotype control: Rabbit IgG-FITC at the same concentration as the SSBP3 antibody to assess non-specific binding

• Negative control: Cells known not to express SSBP3 or with SSBP3 knockdown

• Blocking control: Pre-incubation of the antibody with the immunogen peptide (when available)

• Secondary-only control: For experiments where a secondary antibody is used to amplify the FITC signal

The FITC conjugation has an excitation/emission profile of 499/515 nm, making it compatible with standard FITC filter sets and the 488 nm laser line commonly found in confocal microscopes and flow cytometers .

How can researchers troubleshoot weak or nonspecific signals when using SSBP3 Antibody, FITC conjugated?

When encountering weak or nonspecific signals with SSBP3 Antibody, FITC conjugated, researchers should consider the following troubleshooting approaches:

For weak signals:

• Increase antibody concentration: Try using a higher concentration of the antibody

• Optimize fixation: Different fixation methods (paraformaldehyde, methanol, acetone) can affect epitope accessibility

• Enhance antigen retrieval: For tissue sections or highly cross-linked samples, optimize antigen retrieval methods

• Signal amplification: Consider using anti-FITC secondary antibodies conjugated to brighter fluorophores

• Reduce photobleaching: Minimize exposure to light and use anti-fade mounting media

For nonspecific signals:

• Optimize blocking: Increase blocking time or try different blocking reagents (BSA, serum, commercial blockers)

• Increase washing steps: More thorough or additional washing steps can reduce background

• Reduce antibody concentration: Excessive antibody can increase background

• Cross-adsorbed antibodies: Ensure the antibody has been cross-adsorbed against potential cross-reactive proteins

• Filter samples: For flow cytometry, proper filtering of cell suspensions can reduce aggregates causing false signals

How do experimental conditions affect SSBP3 detection in different cell types?

The detection of SSBP3 can vary significantly across cell types due to differences in expression levels, subcellular localization, and interactions with other proteins. Research indicates that SSBP3 is expressed in various tissues including pancreatic β-cells and embryonic stem cells .

Key considerations for experimental design across different cell types include:

• Expression level variation: SSBP3 expression varies across cell types, necessitating optimization of antibody concentration for each cell type

• Subcellular localization: As a transcription regulator, SSBP3 is predominantly nuclear but may show different localization patterns depending on:

  • Cell cycle stage

  • Differentiation status

  • Activation of specific signaling pathways

• Co-expression with binding partners: Detection efficiency may be affected by the presence of SSBP3 binding partners like Ldb1 and Isl1, which could potentially mask epitopes

• Fixation sensitivity: Different cell types may require distinct fixation protocols to optimally preserve SSBP3 epitopes while maintaining cellular morphology

• Cell-specific autofluorescence: Some cell types (particularly those rich in NADH, flavins, or lipofuscin) exhibit higher autofluorescence in the green spectrum, potentially interfering with FITC detection

When transitioning between cell types, it is advisable to first establish positive controls using cell lines known to express high levels of SSBP3, such as βTC-3 cells or embryonic stem cells , before proceeding to cell types with unknown expression levels.

How should researchers differentiate between specific SSBP3 signals and background in co-localization studies?

In co-localization studies involving SSBP3 Antibody, FITC conjugated, differentiating specific signals from background requires rigorous analytical approaches:

• Quantitative co-localization metrics:

  • Calculate Pearson's correlation coefficient (values between -1 and +1)

  • Determine Manders' overlap coefficient (proportion of SSBP3 signal overlapping with partner protein)

  • Assess spatial intensity correlation analyses

• Single-labeled controls:

  • Perform parallel experiments with each antibody individually to assess bleed-through

  • Use spectral unmixing algorithms if bleed-through cannot be eliminated optically

• Biological validation:

  • Compare co-localization patterns in SSBP3 knockdown or overexpression conditions

  • Examine co-localization following treatment with agents known to affect SSBP3 interactions (such as factors that alter Ldb1-Isl1 complex formation)

• Resolution considerations:

  • Standard confocal microscopy has a resolution limit of ~200nm

  • For more precise co-localization, consider super-resolution techniques like STED, PALM, or STORM

• 3D analysis:

  • Analyze co-localization in all three dimensions rather than in single optical sections

  • Use specialized software (ImageJ with JACoP plugin, Imaris, etc.) for volumetric co-localization analysis

When specifically studying SSBP3's co-localization with known interaction partners like Ldb1 and Isl1, researchers should be aware that these proteins exist in large complexes as demonstrated by sucrose sedimentation data , which may affect the resolution needed to accurately assess co-localization.

What are the expected results when using SSBP3 Antibody, FITC conjugated in different experimental contexts?

When using SSBP3 Antibody, FITC conjugated across different experimental approaches, researchers should anticipate the following typical results:

In immunofluorescence microscopy:

• Predominantly nuclear localization in most cell types

• Potential punctate pattern corresponding to transcriptional complexes

• Co-localization with transcription factors Ldb1 and Isl1 in pancreatic β-cells

• Possible cytoplasmic signal during specific cellular states or in certain cell types

In flow cytometry:

• Positive population shifts when comparing to isotype controls

• Heterogeneous expression levels across cell populations

• Potential correlation with cell cycle phases or differentiation states

• Higher expression in embryonic stem cells undergoing differentiation toward trophoblast lineage

In ChIP experiments:

• Enrichment at promoter regions of target genes such as MafA and Glp1r in β-cells

• Binding to pyrimidine-rich elements similar to those in the alpha 2(I) collagen promoter

• Co-occupancy with Ldb1 and Isl1 at specific genomic loci

• Dynamic binding patterns that may change with cellular differentiation or response to stimuli

In developmental biology studies:

• Increased detection during embryonic stem cell differentiation toward trophoblast lineage

• Association with changes in the methylation status of developmental genes like Elf5

• Correlation with activation of MAPK/Erk1/2 and TGF-β pathways

Understanding these expected patterns helps researchers validate their results and interpret unexpected findings that may represent novel aspects of SSBP3 biology.

How does FITC conjugation compare to other fluorophores for SSBP3 detection in various applications?

FITC conjugation of SSBP3 Antibody offers specific advantages and limitations compared to other fluorophores:

Application-specific considerations:

• Immunofluorescence microscopy:

  • FITC is suitable for single-color detection but may photobleach during extended imaging

  • When performing multi-color imaging, FITC's emission spectrum may overlap with other green-yellow fluorophores

  • FITC offers good signal-to-noise ratio in fixed cells when proper blocking of autofluorescence is employed

• Flow cytometry:

  • FITC is excited efficiently by the standard 488 nm laser

  • FITC may not be optimal for cells with high autofluorescence

  • For dim antigens, brighter alternatives like PE might be preferable

• Multiplex imaging:

  • FITC can be effectively combined with red (e.g., TRITC, Cy3) and far-red (e.g., Cy5, APC) fluorophores

  • When detecting multiple green targets, spectral unmixing or sequential scanning may be necessary

The choice between FITC and other fluorophores should be based on the specific experimental needs, equipment specifications, and the biological question being addressed.

What are the advantages and limitations of using SSBP3 Antibody, FITC conjugated compared to indirect immunofluorescence methods?

Direct detection using SSBP3 Antibody, FITC conjugated offers distinct advantages and limitations compared to indirect detection methods:

Advantages:

• Simplified workflow: Eliminates the need for secondary antibody incubation and washing steps

• Reduced background: Minimizes potential cross-reactivity from secondary antibodies

• Faster protocol: Typically saves 1-2 hours in experimental time

• Improved multiplexing: Allows for simultaneous detection of multiple targets from the same host species

• Defined stoichiometry: Each primary antibody carries a consistent number of fluorophores

Limitations:

• Signal amplification: Direct conjugation typically provides lower signal intensity compared to indirect methods that allow for multiple secondary antibodies binding each primary antibody

• Flexibility: Cannot change detection system without changing the primary antibody

• Cost efficiency: More expensive for screening multiple antibody clones or dilutions

• Shelf-life considerations: Conjugated antibodies may have shorter shelf-life than unconjugated versions

• Epitope accessibility: The conjugation process may occasionally affect antibody binding to certain epitopes

Recommendations for specific scenarios:

• For abundant targets or when minimizing background is critical: Direct detection with SSBP3-FITC

• For low-abundance targets requiring signal amplification: Indirect detection with unconjugated anti-SSBP3

• For multiplexing with other rabbit antibodies: Direct detection with SSBP3-FITC

• For co-localization studies with Ldb1 and Isl1: Consider directly conjugated antibodies for all targets to minimize cross-reactivity

When studying SSBP3's interaction with Ldb1-Isl1 complexes in pancreatic β-cells , the direct conjugation approach may be particularly valuable for reducing background and enabling precise co-localization analysis.

How might SSBP3 Antibody, FITC conjugated be utilized in emerging single-cell analysis technologies?

SSBP3 Antibody, FITC conjugated has significant potential applications in cutting-edge single-cell analysis platforms:

• Single-cell RNA-seq combined with protein detection (CITE-seq/REAP-seq):

  • SSBP3-FITC could be adapted with DNA barcoding for simultaneous detection of SSBP3 protein levels alongside transcriptome analysis

  • This would allow correlation between SSBP3 protein expression and transcriptional states in heterogeneous populations

  • Particularly valuable for studying SSBP3's role in embryonic stem cell differentiation

• Imaging mass cytometry and Multiplex ion beam imaging (MIBI):

  • SSBP3 antibody could be metal-tagged instead of FITC-conjugated for high-dimensional spatial analysis

  • Would enable simultaneous visualization of SSBP3 with dozens of other proteins in tissue context

  • Useful for examining SSBP3's relationship with Ldb1-Isl1 complexes in intact pancreatic islets

• Live-cell tracking with photoactivatable fluorophores:

  • Development of SSBP3 antibodies with photoactivatable versions of FITC or similar fluorophores

  • Would enable dynamic tracking of SSBP3 localization during cell differentiation or in response to stimuli

  • Could provide insights into the kinetics of SSBP3's role in transcriptional complex formation

• Microfluidic antibody capture techniques:

  • Integration of SSBP3-FITC in microfluidic platforms for capturing rare cell types based on SSBP3 expression

  • Would facilitate enrichment of specific cellular subpopulations for downstream analysis

  • Potentially valuable for isolating cells at specific stages of trophoblast differentiation

The adaptation of SSBP3-FITC for these emerging technologies would require additional validation and possibly modification of the antibody or conjugation chemistry, but would significantly expand our understanding of SSBP3's functions at the single-cell level.

What new insights might be gained by studying SSBP3 expression across different developmental stages and disease states?

Investigating SSBP3 expression patterns across developmental trajectories and in disease contexts using the SSBP3 Antibody, FITC conjugated could reveal:

Developmental insights:

• Embryonic development: Given SSBP3's role in trophoblast differentiation , studying its expression during early embryogenesis could reveal critical windows for lineage commitment

• Pancreatic development: As SSBP3 interacts with Ldb1-Isl1 in mature β-cells , tracking its expression during pancreatic organogenesis may identify key developmental transitions

• Stem cell differentiation dynamics: Temporal analysis of SSBP3 expression during directed differentiation protocols could help optimize regenerative medicine approaches

Disease-related applications:

• Diabetes research: Given SSBP3's role in pancreatic β-cell gene regulation , examining its expression in models of diabetes might reveal dysregulation contributing to disease pathogenesis

• Cancer biology: Investigating SSBP3 expression in various cancers, particularly those with aberrant differentiation programs, could identify novel roles in malignancy

• Placental disorders: Since SSBP3 influences trophoblast differentiation , studying its expression in placental pathologies might uncover mechanisms of pregnancy complications

Methodological approaches:

• Tissue microarrays: Analyzing SSBP3 expression across multiple tissues and disease states simultaneously

• Longitudinal sampling: Tracking SSBP3 expression at defined timepoints during development or disease progression

• Single-cell resolution studies: Identifying rare cell populations with distinct SSBP3 expression patterns that might be missed in bulk analyses

• Correlation with epigenetic marks: Given SSBP3's effect on Elf5 promoter methylation , parallel analysis of DNA methylation and SSBP3 binding could reveal mechanisms of epigenetic regulation