SSX2IP Antibody, FITC conjugated

What is SSX2IP and what is its biological significance?

SSX2IP (Synovial Sarcoma X breakpoint-interacting protein 2) is a novel centriolar satellite protein that plays a critical role in the assembly of primary cilia. It localizes to the basal body of primary cilia in both human and murine ciliated cells. Functionally, SSX2IP is essential for the efficient recruitment of the ciliopathy-associated satellite protein Cep290 to both satellites and the basal body . The protein belongs to an adhesion system involved in organizing homotypic, interneuronal, and heterotypic cell-cell adherens junctions (AJs). SSX2IP may connect the nectin-afadin and E-cadherin-catenin systems through alpha-actinin and might participate in organizing the actin cytoskeleton at AJs . Additionally, it functions as a centrosome maturation factor, likely by maintaining the integrity of the pericentriolar material and facilitating proper microtubule nucleation at mitotic spindle poles .

What is the tissue expression pattern of SSX2IP?

SSX2IP is widely expressed throughout the human body, with expression levels varying significantly across different tissues:

This expression pattern suggests broad biological functions across multiple organ systems, with particular importance in neural tissues .

What are the typical applications for FITC-conjugated SSX2IP antibodies?

FITC-conjugated SSX2IP antibodies can be employed in multiple research applications:

• Immunofluorescence microscopy for localization studies of SSX2IP at the basal body of primary cilia

• Flow cytometry for quantitative analysis of SSX2IP expression in different cell populations

• Immunohistochemistry for tissue-specific expression studies

• Live-cell imaging to track SSX2IP dynamics in relation to cilia formation and function

• Co-localization studies with other centriolar and basal body proteins

When designing experiments with FITC-conjugated antibodies, it's important to note that FITC has an excitation/emission spectrum that virtually overlaps with enhanced GFP (eGFP), making them incompatible for use in the same panel .

How should FITC-conjugated SSX2IP antibodies be stored to maintain optimal activity?

For long-term storage, FITC-conjugated antibodies should be kept at -20°C, away from light to prevent photobleaching of the fluorophore . It's crucial to avoid repeated freeze-thaw cycles, as these can significantly reduce antibody activity and fluorescence intensity. For working solutions, storage at 4°C for no more than 1-2 weeks is recommended. The typical formulation contains PBS with 0.05% sodium azide and 50% glycerol, at pH 7.4, which helps stabilize the antibody . For any research-specific applications, validation of storage conditions may be necessary to ensure consistent experimental results.

What are the optimal protocols for conjugating SSX2IP antibodies with FITC?

Several methods exist for FITC conjugation of antibodies, each with distinct advantages:

Dialysis Labeling Method:
This technique can produce conjugates with optimal fluorescein-to-protein ratios of 5-10 in approximately 2 hours . The procedure involves:

• Purifying antibodies through multiple ammonium sulfate fractionations to obtain gamma globulins of adequate purity

• Conjugating the purified antibodies with FITC using controlled dialysis

• Monitoring the fluorescein-to-protein ratio throughout the process to achieve optimal labeling

Lightning-Link® Technology:
A more rapid approach allows FITC conjugation in under 4 hours with minimal hands-on time (approximately 30 seconds) :

• Add the antibody to the Lightning-Link® reagent vial

• Incubate for the specified time (typically 3-4 hours)

• The resulting conjugate can be used without further purification, offering 100% antibody recovery

When selecting a conjugation method, consider factors such as the quantity of antibody available, required conjugation efficiency, and downstream applications.

How can I optimize fixation methods for detecting SSX2IP in primary cilia using FITC-conjugated antibodies?

The detection of centriolar satellite proteins like SSX2IP in primary cilia requires careful optimization of fixation protocols:

For optimal results when studying SSX2IP localization at the basal body of primary cilia, pre-extraction with 0.1% Triton X-100 prior to fixation may improve signal-to-noise ratio by removing cytoplasmic proteins .

What dilution ranges are appropriate for FITC-conjugated SSX2IP antibodies in different applications?

The optimal dilution of FITC-conjugated SSX2IP antibodies varies by application. Based on data for unconjugated SSX2IP antibodies, the following ranges can serve as starting points :

Each new lot of antibody should be titrated to determine the optimal working dilution for your specific experimental system. For FITC-conjugated antibodies, it's advisable to start with a more concentrated dilution than would be used for unconjugated antibodies, as the conjugation process may slightly reduce binding efficiency.

How does SSX2IP function in the recruitment of ciliary proteins and what methods can be used to study this?

SSX2IP plays a crucial role in recruiting proteins to the primary cilium through multiple mechanisms:

• Cep290 Recruitment: SSX2IP is essential for the efficient recruitment of Cep290 to both centriolar satellites and the basal body. This recruitment function can be studied using small interfering RNA (siRNA) knockdown approaches in human cells .

• BBSome Entry: Loss of SSX2IP drastically reduces entry of the BBSome complex, which functions to target membrane proteins to primary cilia. This effect can be visualized using FITC-conjugated antibodies against BBSome components in SSX2IP-depleted cells .

• Rab8 Accumulation: SSX2IP is required for efficient accumulation of Rab8, a key regulator of ciliary membrane protein targeting. Quantitative immunofluorescence using FITC-conjugated anti-Rab8 antibodies can measure this effect .

• SSTR3 Targeting: SSX2IP knockdown limits the targeting of somatostatin receptor 3 (SSTR3), a ciliary membrane protein and BBSome cargo. This can be assessed through co-localization studies with FITC-labeled antibodies .

Experimental approaches to study these functions include:

• siRNA-mediated knockdown followed by immunofluorescence microscopy

• Rescue experiments with SSX2IP mutants lacking specific domains

• Proximity labeling techniques to identify novel SSX2IP interaction partners

• Live-cell imaging of ciliary protein trafficking using FITC-labeled antibodies

What strategies can minimize spectral overlap when using FITC-conjugated SSX2IP antibodies in multicolor immunofluorescence?

When designing multicolor immunofluorescence panels that include FITC-conjugated SSX2IP antibodies, managing spectral overlap is essential for accurate interpretation of results:

• Strategic fluorophore selection: Choose fluorophores with minimal spectral overlap with FITC. For example:

  • DAPI for nuclear staining (blue)

  • TRITC or Cy3 for red channel

  • Far-red fluorophores like Cy5 or Alexa Fluor 647

• Avoid GFP reporters: As shown in Figure 4 from the search results, eGFP and FITC have virtually overlapping excitation/emission spectra, making them incompatible for use in the same panel . If working with GFP-expressing cells, consider using a different fluorophore for antibody conjugation.

• Sequential imaging: For confocal microscopy, acquire images of each fluorophore sequentially rather than simultaneously to minimize bleed-through.

• Compensation controls: For flow cytometry applications, proper compensation is essential:

  • Use single-stained controls for each fluorophore

  • Apply mathematical compensation to correct for spectral overlap

• Spectral unmixing: Advanced microscopy systems with spectral detection capabilities can mathematically separate overlapping fluorophore signals based on their unique spectral signatures.

How can quantitative analysis of SSX2IP localization be optimized using FITC-conjugated antibodies?

Quantitative analysis of SSX2IP localization using FITC-conjugated antibodies requires rigorous experimental design and image analysis approaches:

• Standardized image acquisition:

  • Use consistent exposure settings between samples

  • Acquire images below saturation

  • Include reference standards for fluorescence intensity calibration

• Background correction methods:

  • Subtract autofluorescence using unstained controls

  • Apply local background subtraction algorithms

  • Use rolling ball background subtraction for uneven backgrounds

• Colocalization analysis with basal body markers:

  • Calculate Pearson's or Mander's coefficients for colocalization with basal body markers

  • Consider distance-based measurements from the center of the basal body

• Quantification of intensity profiles:

  • Generate line profiles through centriolar satellites

  • Measure fluorescence intensity distribution in relation to distance from the centriole

• 3D analysis considerations:

  • Z-stack acquisition with appropriate step size

  • Maximum intensity projections vs. 3D rendering

  • Volume-based quantification of SSX2IP-positive structures

For high-throughput analysis, automated image analysis pipelines can be developed using software like ImageJ/Fiji, CellProfiler, or custom scripts to standardize quantification across multiple experimental conditions.

What are the effects of SSX2IP depletion on ciliary structure and function, and how can these be measured?

SSX2IP depletion has multiple effects on ciliary structure and function that can be quantitatively assessed:

• Axoneme length reduction:
  SSX2IP knockdown significantly reduces axoneme length . This can be measured using:

  • Immunofluorescence with acetylated tubulin (ciliary marker) and FITC-conjugated SSX2IP antibodies

  • Scanning electron microscopy for ultrastructural analysis

  • Time-lapse imaging to track ciliary growth rates

• Ciliary protein mislocalization:
  The loss of SSX2IP disrupts proper localization of multiple ciliary proteins :

  • BBSome components fail to enter cilia

  • Rab8 accumulation is reduced

  • SSTR3 targeting is limited

  These effects can be quantified through colocalization analysis and fluorescence intensity measurements.

• Functional assays:

  • Ciliary signaling: Measure Hedgehog pathway activation through Gli reporter assays

  • Mechanosensation: Calcium imaging in response to fluid flow

  • Chemosensation: cAMP assays in response to GPCR ligands that signal through ciliary receptors

• Rescue experiments:
  Reintroduce wild-type or domain-specific mutants of SSX2IP to identify regions critical for ciliary functions using FITC-conjugated antibodies against ciliary markers.

What are common issues with FITC-conjugated antibodies and how can they be addressed?

When working with FITC-conjugated SSX2IP antibodies, researchers may encounter several technical challenges:

• Photobleaching:

  • Issue: FITC is relatively prone to photobleaching during extended imaging sessions

  • Solution: Add anti-fade reagents to mounting media, minimize exposure time, and consider acquiring FITC channel images first

• Autofluorescence:

  • Issue: Tissues and fixatives can generate autofluorescence in the FITC channel

  • Solution: Include unstained controls, use Sudan Black B (0.1-0.3%) to quench autofluorescence, or consider spectral unmixing

• pH sensitivity:

  • Issue: FITC fluorescence intensity is optimal at pH 8.0 and decreases at lower pH

  • Solution: Ensure buffers are maintained at pH 7.5-8.0 for maximum fluorescence

• Over-conjugation:

  • Issue: Excessive FITC molecules per antibody can cause self-quenching and reduced antibody affinity

  • Solution: Use conjugates with optimal fluorescein-to-protein ratios of 5-10

• Non-specific binding:

  • Issue: High background due to non-specific binding

  • Solution: Optimize blocking (3-5% BSA or serum), include proper controls, and validate antibody specificity

What quality control measures should be implemented when using FITC-conjugated SSX2IP antibodies?

Rigorous quality control is essential for generating reliable data with FITC-conjugated SSX2IP antibodies:

• Validation controls:

  • Positive control: Tissues/cells known to express SSX2IP (brain, kidney, or cultured ciliated cells)

  • Negative control: SSX2IP knockdown cells or tissues with minimal expression (pancreas, spleen)

  • Isotype control: FITC-conjugated non-specific IgG of the same host species

  • Absorption control: Pre-incubation of antibody with immunizing peptide

• Conjugation quality assessment:

  • Measure protein concentration and fluorophore absorbance to calculate the fluorophore-to-protein ratio

  • Verify that the F/P ratio falls within the optimal range of 5-10

  • Assess antibody functionality post-conjugation through activity assays

• Batch consistency:

  • Test each new lot against a reference standard

  • Document lot-specific optimal dilutions and staining patterns

  • Consider preparing large batches of working dilutions to minimize inter-experimental variation

• Specificity verification:

  • Western blot to confirm single band at expected molecular weight

  • Immunoprecipitation followed by mass spectrometry

  • Competitive binding assays with unconjugated antibody

How are FITC-conjugated SSX2IP antibodies advancing our understanding of ciliopathies?

FITC-conjugated SSX2IP antibodies have become valuable tools for investigating ciliopathies, a diverse group of genetic disorders resulting from ciliary dysfunction. SSX2IP's critical role in ciliary assembly and protein recruitment makes it an important target for understanding disease mechanisms .

Recent research has established SSX2IP as a novel targeting factor for ciliary membrane proteins that cooperates with multiple key ciliary components:

• Cep290, a protein mutated in several ciliopathies including Joubert syndrome and Bardet-Biedl syndrome

• The BBSome complex, implicated in Bardet-Biedl syndrome

• Rab8, a small GTPase essential for ciliary membrane protein trafficking

By utilizing FITC-conjugated SSX2IP antibodies in high-resolution imaging studies, researchers have visualized the precise localization of SSX2IP at the basal body and tracked its dynamics during cilia formation and maintenance . This has provided insights into the molecular mechanisms underlying ciliopathies and potential therapeutic targets.

Future research directions may include:

• Structural studies of SSX2IP interactions with ciliary targeting complexes

• Development of small molecule modulators of SSX2IP function

• Application of super-resolution microscopy with FITC-conjugated SSX2IP antibodies to reveal nanoscale organization of ciliary transition zone

What emerging methodologies might enhance research using FITC-conjugated SSX2IP antibodies?

Several cutting-edge technologies are poised to enhance research applications of FITC-conjugated SSX2IP antibodies:

• Super-resolution microscopy:
  Techniques such as STORM, PALM, and SIM can overcome the diffraction limit of conventional fluorescence microscopy, enabling visualization of SSX2IP organization at nanometer resolution.

• Expansion microscopy:
  This approach physically expands specimens using swellable polymers, providing an alternative route to super-resolution imaging of SSX2IP localization in ciliary structures.

• Live-cell ciliary protein tracking:
  Combining FITC-conjugated SSX2IP antibody fragments with cell-penetrating peptides may enable real-time tracking of SSX2IP dynamics in living cells.

• Proximity labeling:
  BioID or APEX2 fusions with SSX2IP can identify novel protein interactions at centriolar satellites and the basal body, complementing immunofluorescence studies.

• Organoid models:
  FITC-conjugated SSX2IP antibodies can be applied to study ciliary biology in organoid models of development and disease, offering more physiologically relevant contexts than traditional cell culture.

• Multiplexed imaging: Cyclic immunofluorescence or mass cytometry approaches can overcome the spectral limitations of conventional fluorescence microscopy, allowing simultaneous detection of dozens of proteins along with FITC-conjugated SSX2IP antibodies.