SUN2 Antibody, FITC conjugated

What is SUN2 protein and why is it an important research target?

SUN2 (Sad1 and UNC-84 domain containing 2) is an inner nuclear membrane (INM) protein with a molecular weight of approximately 80-85 kDa. It functions as a critical component of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, which connects the nuclear lamina to the cytoskeleton. SUN2 contains a C-terminal SUN domain that extends into the perinuclear space, where it interacts with KASH domain proteins in the outer nuclear membrane.

Research on SUN2 is particularly important because it plays crucial roles in maintaining nuclear architecture, positioning the nucleus within cells, and facilitating chromatin organization. Recent studies have shown that SUN2 is regulated by a balance between phosphorylation by Casein Kinase 2 and dephosphorylation by C-terminal domain Nuclear Envelope Phosphatase 1 (CTDNEP1) . This phosphorylation balance controls SUN2 binding to the ubiquitin ligase SCFβTrCP, which promotes its ubiquitination, leading to extraction from the membrane by the AAA ATPase p97 and subsequent proteasomal degradation .

Importantly, abnormal accumulation of SUN2 results in aberrant nuclear architecture, increased vulnerability to DNA damage, and chromosomal segregation defects during mitosis, making it a valuable target for research into nuclear envelope dynamics and genomic stability .

What are the optimal applications for FITC-conjugated SUN2 antibodies?

FITC-conjugated SUN2 antibodies are particularly valuable for immunofluorescence (IF) applications, including both fixed-cell immunocytochemistry (ICC) and tissue immunohistochemistry (IHC). These applications typically require dilutions ranging from 1:500 to 1:2000, though this may vary based on the specific antibody preparation and experimental conditions .

For immunofluorescence experiments, FITC-conjugated SUN2 antibodies enable direct visualization of SUN2 localization without requiring secondary antibody incubation steps, saving time and reducing potential background. The green fluorescence of FITC (excitation ~495 nm, emission ~519 nm) is compatible with most standard fluorescence microscopy filter sets and allows for multiplexing with other fluorophores that emit in different wavelength ranges.

The direct conjugation approach is particularly useful in co-localization studies where primary antibodies from the same species would otherwise create cross-reactivity issues with secondary antibodies. FITC-conjugated antibodies also perform well in flow cytometry applications for quantifying SUN2 expression in cell populations.

What are the recommended storage and handling procedures for FITC-conjugated antibodies?

FITC-conjugated antibodies require special handling to maintain optimal fluorescence and binding activity. Based on standard protocols for fluorophore-conjugated antibodies, the following practices are recommended:

• Storage conditions: Store at -20°C in a light-protected container. Most FITC-conjugated antibodies are supplied in a buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Light protection: FITC is highly susceptible to photobleaching. Always protect the antibody from light exposure during storage and handling . Use amber tubes or wrap containers in aluminum foil.

• Aliquoting: For antibodies stored at -20°C, aliquoting is generally unnecessary, though it may be advisable for frequently used antibodies to prevent freeze-thaw cycles .

• Stability: When properly stored, FITC-conjugated antibodies typically remain stable for one year after shipment .

• Working solution preparation: When preparing dilutions, use freshly made buffers. For immunofluorescence applications, dilution in PBS containing 10% fetal bovine serum is typically recommended .

• Azide precautions: Note that sodium azide, commonly included as a preservative, is toxic and can form explosive compounds with heavy metals in plumbing systems . Always dispose of azide-containing solutions according to local regulations.

What are the recommended dilution ratios for different experimental applications?

The optimal dilution ratios for FITC-conjugated SUN2 antibodies vary by application:

It is strongly recommended that each laboratory empirically determines the appropriate dilution for their specific experimental conditions and sample types . Titration experiments should be performed to identify the optimal antibody concentration that provides maximum specific signal with minimal background.

How can FITC-conjugated SUN2 antibodies be used to study nuclear envelope dynamics during mitosis?

Studying nuclear envelope dynamics during mitosis using FITC-conjugated SUN2 antibodies requires careful experimental design to capture the dynamic reorganization of the nuclear membrane. Here's a methodological approach:

• Live-cell imaging: For dynamic studies, cells can be transfected with a low-concentration SUN2-GFP construct to monitor movement in real-time, with FITC-conjugated antibodies used in parallel fixed-cell experiments at various time points for validation.

• Cell synchronization: Use nocodazole or thymidine blocking to synchronize cells at specific cell cycle stages, then release and fix cells at precise time intervals during mitotic progression.

• Co-staining protocol: Combine FITC-conjugated SUN2 antibodies with markers for:

  • Chromatin (DAPI or Hoechst)

  • Nuclear lamina (Lamin B1)

  • Mitotic spindle (α-tubulin)

  • Cell cycle markers (phospho-histone H3)

• Quantification approach: Measure the intensity and distribution of SUN2 signal around chromatin at different mitotic stages. Tracking SUN2 redistribution is particularly important as research has shown that abnormal SUN2 levels are associated with increased lagging chromosomes during mitosis .

• Super-resolution microscopy: Techniques like structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) can reveal detailed spatial organization of SUN2 during nuclear envelope breakdown and reassembly.

A key research finding is that accumulation of non-degradable forms of SUN2 results in aberrant nuclear architecture and mitotic defects . Use this assay to examine how manipulations that alter SUN2 degradation (such as inhibiting the proteasome or p97 ATPase) affect nuclear envelope dynamics during mitosis.

What controls should be included when using FITC-conjugated SUN2 antibodies in DNA damage studies?

When designing experiments to study the relationship between SUN2 and DNA damage responses using FITC-conjugated antibodies, include the following controls and considerations:

• Essential negative controls:

  • Isotype control: Use a FITC-conjugated antibody of the same isotype but with irrelevant specificity

  • Secondary antibody only (if using indirect FITC detection)

  • Untreated cells (without DNA damage induction)

• Positive controls:

  • γH2AX staining as a marker of DNA double-strand breaks

  • Known DNA damage-inducing agents (e.g., etoposide, doxorubicin, or ionizing radiation) with established time-response relationships

• Experimental validation controls:

  • SUN2 knockdown/knockout cells to confirm antibody specificity

  • Cells expressing SUN2 mutants that cannot be degraded, which have been shown to increase vulnerability to DNA damage

  • Treatment with proteasome inhibitors to prevent SUN2 degradation

• Technical considerations:

  • Use sequential scanning in confocal microscopy to prevent bleed-through between fluorescence channels

  • Include calibration standards to normalize fluorescence intensity between experiments

  • Perform time-course experiments to capture both early and late DNA damage responses

• Data analysis recommendations:

  • Quantify co-localization between SUN2 and DNA damage markers

  • Measure nuclear morphology parameters (as aberrant nuclear architecture correlates with SUN2 accumulation)

  • Analyze repair kinetics by measuring resolution of DNA damage foci over time

Recent research has shown that non-degradable SUN2 results in vulnerability to DNA damage , suggesting that proper regulation of SUN2 levels is crucial for genome stability. These controls will help establish whether observed phenotypes are specifically related to SUN2 function.

What might cause variability in fluorescence intensity when using FITC-conjugated SUN2 antibodies?

Variability in fluorescence intensity is a common challenge in immunofluorescence experiments using FITC-conjugated antibodies. Several factors may contribute to this issue:

• Photobleaching effects: FITC is particularly susceptible to photobleaching under continuous light exposure . To minimize this:

  • Limit exposure to excitation light during image acquisition

  • Use anti-fade mounting media containing agents like DABCO or propyl gallate

  • Consider acquiring images from unexposed fields first

  • Use identical exposure settings for all comparable samples

• Fixation and permeabilization variables:

  • Different fixatives (paraformaldehyde, methanol, etc.) affect epitope accessibility

  • Overfixation can mask epitopes and reduce signal

  • Insufficient permeabilization limits antibody access to nuclear antigens like SUN2

  • Solution: Test multiple fixation protocols in parallel to optimize for SUN2 detection

• Biological variation in SUN2 expression:

  • SUN2 levels are regulated by phosphorylation, ubiquitination, and proteasomal degradation

  • Cell cycle stage influences SUN2 detection, with potential redistribution during mitosis

  • Expression levels vary between cell types and experimental conditions

• Technical variables:

  • Antibody lot-to-lot variation

  • Storage conditions affecting FITC conjugate stability

  • Inconsistent washing steps leading to variable background

  • Suboptimal antibody concentration (requires titration for each application)

• Quantification approach:

  • Include internal standards in each experiment

  • Normalize fluorescence intensity to cell number or nuclear area

  • Use automated image analysis with consistent thresholding parameters

Importantly, SUN2 has been shown to undergo regulated degradation , meaning its levels can fluctuate based on cellular conditions. Consider including phosphatase inhibitors in your sample preparation if studying phosphorylation-dependent aspects of SUN2 biology.

How can digital image analysis be optimized for experiments using FITC-conjugated SUN2 antibodies?

Optimizing digital image analysis for FITC-conjugated SUN2 antibody experiments requires addressing both acquisition and analysis parameters:

• Acquisition optimization:

  • Use the same microscope settings (exposure time, gain, offset) for all comparable samples

  • Ensure that pixel intensities are not saturated at the upper end of the dynamic range

  • Acquire z-stacks when analyzing nuclear envelope proteins to capture the complete 3D structure

  • Include reference standards in each imaging session to normalize between experiments

• Preprocessing steps:

  • Apply flat-field correction to compensate for uneven illumination

  • Use deconvolution algorithms to improve signal-to-noise ratio

  • Implement background subtraction methods appropriate for nuclear envelope signals

• Nuclear envelope segmentation strategies:

  • Develop specific algorithms for nuclear envelope detection (ring-like structures)

  • Use nuclear stains (DAPI/Hoechst) to identify nuclei, then create nuclear envelope masks

  • Implement specialized edge detection algorithms for capturing the thin nuclear envelope signal

• Quantification parameters for SUN2 analysis:

  • Mean intensity along the nuclear envelope

  • Coefficient of variation to measure distribution homogeneity

  • Nuclear envelope-to-nucleoplasm ratio to assess SUN2 localization

  • Nuclear morphology metrics (circularity, size, aspect ratio) to correlate with SUN2 distribution

• Statistical approaches:

  • Analyze sufficient cell numbers (typically >100 cells per condition)

  • Use hierarchical statistical models that account for cell-to-cell variability

  • Apply appropriate tests for non-normally distributed data (common in fluorescence intensity measurements)

• Advanced analysis for SUN2 biology:

  • Develop co-localization metrics with other nuclear envelope proteins

  • Implement temporal analysis tools for time-lapse experiments

  • Create analytical pipelines to correlate SUN2 levels with nuclear deformities, which have been linked to SUN2 accumulation

Software packages like CellProfiler, ImageJ/FIJI with nuclear envelope analysis plugins, or custom Python/MATLAB scripts can be adapted for these specialized analyses. Document all image processing steps thoroughly to ensure reproducibility.

How can FITC-conjugated SUN2 antibodies be used to investigate chromatin-nuclear envelope interactions?

Investigating chromatin-nuclear envelope interactions using FITC-conjugated SUN2 antibodies requires specialized approaches that bridge cytological observation with molecular mechanism:

• Chromatin immunoprecipitation (ChIP) followed by immunofluorescence:

  • Perform ChIP using antibodies against chromatin marks or specific genomic regions

  • Follow with immunofluorescence using FITC-conjugated SUN2 antibodies

  • This combination allows identification of specific chromatin regions that interact with SUN2

• 3D immuno-FISH approach:

  • Use fluorescence in situ hybridization (FISH) to label specific genomic loci

  • Combine with FITC-conjugated SUN2 immunofluorescence

  • Analyze spatial relationships between labeled genomic regions and the nuclear envelope

• Proximity ligation assay (PLA) adaptation:

  • Combine FITC-conjugated SUN2 antibodies with antibodies against chromatin proteins

  • Use PLA to detect close interactions (<40 nm)

  • Quantify interaction frequency in different cellular states

• Biochemical fractionation validation:

  • Isolate nuclear envelope fractions

  • Analyze associated chromatin by sequencing or mass spectrometry

  • Use results to guide targeted immunofluorescence with FITC-conjugated SUN2 antibodies

• Perturbation experiments:

  • Express non-degradable SUN2 mutants, which have been shown to cause aberrant nuclear architecture

  • Analyze changes in chromatin organization and gene expression

  • Use FITC-conjugated SUN2 antibodies to confirm protein accumulation at the nuclear envelope

• Advanced microscopy applications:

  • Implement live super-resolution microscopy with complementary tagged chromatin components

  • Use FRET (Fluorescence Resonance Energy Transfer) between FITC-SUN2 and chromatin proteins tagged with appropriate acceptor fluorophores

  • Apply correlative light and electron microscopy (CLEM) to combine fluorescence localization with ultrastructural details

These approaches can help elucidate how SUN2's degradation pathway, which involves ubiquitination and extraction from the membrane by p97 ATPase , influences chromatin organization and gene expression programs.

What techniques can be used to quantify SUN2 protein levels using FITC-conjugated antibodies?

Multiple techniques can be employed to quantify SUN2 protein levels using FITC-conjugated antibodies, each with specific advantages for different research questions:

• Flow cytometry quantification:

  • Provides population-level analysis of SUN2 expression

  • Allows simultaneous analysis of multiple parameters (cell cycle, apoptosis)

  • Protocol adaptation: Fixation and permeabilization must ensure nuclear envelope accessibility

  • Calibration: Use quantitative fluorescence calibration beads to convert fluorescence to molecules of equivalent soluble fluorochrome (MESF)

  • Applications: Particularly useful for examining how SUN2 levels change in response to treatments affecting its degradation pathway

• Quantitative immunofluorescence microscopy:

  • Preserves spatial information about SUN2 distribution

  • Applicable techniques include wide-field, confocal, or super-resolution microscopy

  • Calibration: Include internal standards with known FITC concentrations

  • Analysis approach: Measure integrated fluorescence intensity along the nuclear envelope

  • Normalization strategies: Use nuclear area or DNA content for cell-to-cell comparisons

• Microplate reader-based assays:

  • High-throughput quantification suitable for screening applications

  • Protocol: Fix cells in microplates, immunostain with FITC-conjugated SUN2 antibodies

  • Controls: Include calibration wells with known quantities of FITC

  • Advantages: Rapid analysis of multiple conditions simultaneously

  • Limitations: Loses spatial information about SUN2 distribution

• In-cell western techniques:

  • Combines the quantitative aspects of western blotting with in situ detection

  • Allows normalization to housekeeping proteins or total protein

  • Enables direct comparison between multiple treatment conditions

  • Analysis: Use infrared imaging systems capable of detecting FITC signal

• Image cytometry platforms:

  • Combines high-throughput imaging with automated quantification

  • Ideal for experiments requiring both population statistics and spatial information

  • Can correlate SUN2 levels with nuclear morphology parameters

  • Particularly useful for studying how non-degradable SUN2 affects nuclear architecture

• Sample preparation considerations:

  • Include phosphatase inhibitors if studying phosphorylation-dependent regulation of SUN2

  • Consider proteasome inhibitors to block SUN2 degradation for baseline measurements

  • Use p97 ATPase inhibitors to prevent extraction of ubiquitinated SUN2 from the membrane

These quantitative approaches enable precise measurement of SUN2 levels in various experimental contexts, facilitating research into its regulated degradation and role in maintaining nuclear architecture.