SND1 Antibody, FITC conjugated

What is SND1 and why is it an important research target?

SND1 is a multifunctional protein also known as p100, TSN, and TDRD11 that contains four tandem Staphylococcal nuclease-like (SN) domains and a C-terminal Tudor domain that interrupts a fifth SN domain . It functions as a "reader" protein that recognizes symmetric dimethylarginine (SDMA) marks through its Tudor domain, particularly those deposited by protein arginine methyltransferase 5 (PRMT5) . SND1 is particularly enriched in secretory tissues like the liver and pancreas .

SND1 has gained significant research attention because it is upregulated in several cancers and positively correlates with worse disease prognosis . For instance, 74% of hepatocellular carcinoma patient samples show SND1 overexpression . In gliomas, SND1 serves as an independent predictor of poor prognosis and facilitates proliferation, migration, and invasion of glioma cells through its function as a novel chromatin architectural modifier .

What cellular components does SND1 antibody typically detect?

SND1 antibody detects the SND1 protein primarily in the nucleus and cytoplasm, reflecting its multifunctional role in cellular processes. Western blot analysis has confirmed positive detection in multiple cell lines including A549, Jurkat, COLO 320, and HeLa cells . Immunohistochemistry has successfully detected SND1 in human breast hyperplasia tissue, breast cancer tissue, and liver tissue . Immunofluorescence studies have also validated SND1 detection in HeLa cells .

How does FITC conjugation affect SND1 antibody applications?

When conjugated with FITC (Fluorescein isothiocyanate), SND1 antibody emits green fluorescence (excitation ~495 nm, emission ~520 nm), making it suitable for direct detection in immunofluorescence microscopy and flow cytometry without requiring secondary antibodies. This conjugation provides advantages for multicolor immunofluorescence experiments, reduces background signal, and simplifies experimental workflows by eliminating secondary antibody incubation steps.

What are the recommended storage conditions for SND1 antibody, FITC conjugated?

FITC-conjugated antibodies are typically light-sensitive and should be stored in dark conditions at 2-8°C for short-term storage (1-2 weeks). For long-term storage, aliquoting and freezing at -20°C or -80°C is recommended to prevent repeated freeze-thaw cycles that may damage the antibody and reduce FITC fluorescence intensity. Diluted working solutions should be prepared fresh and used within the same day for optimal results.

How does SND1's Tudor domain function influence experimental design when using SND1 antibody?

The Tudor domain of SND1 forms a four-residue aromatic cage involving F740, Y746, Y763, and Y766 that mediates binding to SDMA-containing peptides . This domain is critical for SND1's function as a reader of methylarginine marks. When designing experiments with SND1 antibody, researchers should consider whether the antibody's epitope includes or affects the Tudor domain.

For functional studies examining SND1's methylarginine reader role, it's crucial to verify that the antibody does not interfere with Tudor domain interactions. Experiments comparing wild-type SND1 with Tudor domain mutants (such as Y766L) have demonstrated that mutations in the aromatic cage dramatically reduce binding to SDMA-containing peptides . When using SND1 antibody in proximity ligation assays or co-immunoprecipitation studies to investigate Tudor domain-dependent interactions, careful validation is required to ensure the antibody doesn't disrupt these interactions.

What are the methodological considerations for using SND1 antibody, FITC conjugated in multi-parameter flow cytometry?

When incorporating FITC-conjugated SND1 antibody in multi-parameter flow cytometry, researchers must carefully plan the antibody panel to avoid spectral overlap. Since FITC emits in the green spectrum, other fluorophores with minimal spectral overlap (such as PE, APC, or BV421) should be selected for additional markers. Proper compensation controls are essential, including single-stained controls for each fluorochrome and an unstained control.

For intracellular SND1 detection, fixation and permeabilization protocols must be optimized, as excessive fixation may mask epitopes while insufficient permeabilization may limit antibody access. When studying SND1 in cancer cells like glioma or hepatocellular carcinoma, consider including markers for cell proliferation (such as Ki-67) to correlate with SND1 expression, as studies have demonstrated a relationship between SND1 levels and cellular proliferation .

How can chromatin immunoprecipitation (ChIP) experiments with SND1 antibody reveal SND1's role in chromatin architecture?

Research has shown that SND1 functions as a chromatin architectural modifier that can facilitate malignant glioma phenotypes by epigenetically inducing chromatin topological interaction . When designing ChIP experiments with SND1 antibody, several factors should be considered to effectively investigate this function.

First, crosslinking conditions should be optimized for chromatin architecture studies, potentially using dual crosslinking with both formaldehyde and protein-protein crosslinkers. ChIP-chip or ChIP-seq analysis has identified SND1 binding to the promoters of approximately 2505 genes in U118MG glioma cells . When analyzing ChIP data, correlation with gene expression data is valuable—previous studies combined ChIP results with mRNA expression analysis to identify direct and indirect SND1 targets .

For advanced studies, consider combining SND1 ChIP with chromosome conformation capture (3C) techniques to directly investigate SND1's role in chromatin topology, as research has shown that SND1 can remodel chromatin conformation at specific promoters such as RhoA .

What are the optimal protocols for immunofluorescence staining using SND1 antibody, FITC conjugated?

For immunofluorescence applications with FITC-conjugated SND1 antibody, use the following optimized protocol:

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 for 10 minutes

• Block with 1-5% BSA or normal serum for 1 hour

• Incubate with FITC-conjugated SND1 antibody at 1:50-1:500 dilution (optimize based on application)

• Wash thoroughly with PBS

• Counterstain nuclei with DAPI

• Mount using anti-fade mounting medium

For tissue sections, antigen retrieval may be necessary. Published protocols recommend using TE buffer (pH 9.0) or alternatively citrate buffer (pH 6.0) for optimal SND1 detection in tissues like breast cancer and liver samples .

How can SND1 antibody be validated for specificity in glioma and hepatocellular carcinoma models?

To validate SND1 antibody specificity in cancer models, a multi-approach validation strategy should be employed:

• Western blot validation: Compare antibody detection in control versus SND1 knockdown cells (using SND1-sh1, SND1-sh2 as described in glioma studies) . Expected results should show reduced signal in knockdown samples.

• Genetic models validation: Utilize the available SND1 knockout (Snd1 KO) and Tudor domain mutant (Snd1 KI) mouse models for antibody validation . This approach helps distinguish between complete loss of SND1 and specific Tudor domain dysfunction.

• Immunohistochemistry correlation: For glioma studies, perform parallel staining with SND1 antibody and Ki-67 (MKI67), as their expression has been shown to correlate in glioma specimens of different WHO grades .

• Peptide competition assay: Pre-incubate the antibody with a blocking peptide containing the epitope sequence to confirm specificity.

What are the considerations for quantitative analysis of SND1 expression using FITC-conjugated antibody in flow cytometry?

For quantitative flow cytometry analysis of SND1 expression:

• Standardization: Use calibration beads with known quantities of fluorochrome to convert fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF) for consistent quantification across experiments.

• Controls: Include isotype controls conjugated with FITC at the same concentration to account for non-specific binding. For glioma studies, consider using cell lines with known SND1 expression levels (U118MG or patient-derived primary GBM cells) as positive controls .

• Gating strategy: As SND1 expression varies with cell cycle and may correlate with proliferation markers, consider using DNA content staining with far-red fluorochromes (to avoid FITC overlap) for cell cycle analysis in conjunction with SND1 staining.

• Signal stability: FITC is prone to photobleaching and pH sensitivity. Maintain consistent pH in buffers (ideally pH 7.4-8.0 for optimal FITC fluorescence) and minimize light exposure during sample processing.

How to troubleshoot weak signal when using SND1 antibody, FITC conjugated?

When encountering weak signal with FITC-conjugated SND1 antibody, consider these troubleshooting steps:

• Antibody concentration: Increase antibody concentration within recommended range (1:50-1:500) . Titration experiments should be performed to determine optimal concentration for each application.

• Antigen retrieval optimization: For tissue sections, test different antigen retrieval methods. Research indicates that TE buffer (pH 9.0) may provide better results than citrate buffer (pH 6.0) for SND1 detection .

• Fixation assessment: Overfixation can mask epitopes. If using formalin-fixed samples, reduce fixation time or test alternative fixatives like methanol-acetone.

• Signal amplification: For very low expression samples, consider using anti-FITC secondary antibodies conjugated to brighter fluorophores or enzyme systems.

• Fresh antibody aliquots: FITC conjugates lose fluorescence intensity over time and with repeated freeze-thaw cycles. Use freshly thawed aliquots for critical experiments.

How to interpret SND1 antibody staining patterns in relation to cancer progression?

Interpreting SND1 staining patterns requires consideration of several factors based on published research:

What experimental controls are essential when investigating SND1's role in chromatin architecture using FITC-conjugated antibody?

When studying SND1's chromatin architectural functions, these controls are essential:

• Tudor domain mutant controls: Include experiments with SND1 Tudor domain mutants (Y766L) that disrupt SDMA binding to distinguish between Tudor-dependent and independent functions .

• SND1 knockdown validation: Use established SND1 shRNA constructs (SND1-sh1, SND1-sh2) and rescue expression (SND1-sh1/SND1, SND1-sh2/SND1) as controls to confirm antibody specificity and validate phenotypic effects .

• Chromatin conformation controls: When investigating SND1's role in chromatin topology, include controls for general chromatin accessibility (such as ATAC-seq) to distinguish between specific SND1-mediated conformational changes and global chromatin alterations.

• Target gene expression controls: Monitor expression of known SND1 target genes (RhoA, CCND1, CCNE1, CDK4, and CDKN1B) to validate functional consequences of SND1-mediated chromatin alterations .

How might SND1 antibodies contribute to developing targeted therapies for glioma and hepatocellular carcinoma?

SND1 antibodies could contribute to therapeutic development through several approaches:

• Target validation: FITC-conjugated SND1 antibodies can help validate SND1 as a therapeutic target by visualizing its expression in patient-derived xenografts before and after experimental treatments.

• Companion diagnostics: Given that SND1 overexpression is an independent predictor of poor prognosis in glioma patients , SND1 antibodies could be developed into companion diagnostic tools to identify patients who might benefit from SND1-targeted therapies.

• Therapeutic antibody development: While the current FITC-conjugated antibodies are research tools, they provide a foundation for developing therapeutic antibodies targeting SND1, potentially disrupting its chromatin remodeling functions in cancer cells.

• Drug screening platforms: High-content screening using SND1 antibodies could identify compounds that modulate SND1 expression or alter its subcellular localization, potentially identifying new therapeutic candidates.

What are the implications of SND1's Tudor domain function for experimental design in epigenetic research?

The Tudor domain's role as a reader of SDMA marks has significant implications for epigenetic research design:

• Crosstalk with PRMT5 inhibition: As SND1 recognizes methylation marks deposited by PRMT5 , experiments with PRMT5 inhibitors should include analysis of SND1 localization and function to understand the complete mechanism of action.

• Epigenetic reader-writer networks: Studies investigating SND1 should consider the broader context of epigenetic regulation, including potential redundancy or compensation by other Tudor domain-containing proteins.

• Methylarginine interactome studies: FITC-conjugated SND1 antibodies can be used in proximity ligation assays to map the network of proteins that interact with SND1 in a methylarginine-dependent manner, providing insights into its reader function in different cellular contexts.

• Therapeutic targeting strategy: The finding that both complete SND1 knockout and specific Tudor domain mutation confer resistance to carcinogen-induced hepatocellular carcinoma suggests multiple possible approaches for therapeutic intervention, which can be monitored using appropriate antibodies.