ISYNA1 Antibody

What are the recommended dilutions for ISYNA1 antibody applications?

When working with ISYNA1 antibodies, optimizing antibody dilutions is critical for experimental success. Based on validated protocols, the following dilutions are recommended for common applications:

It's important to note that optimal dilutions may vary based on the specific antibody clone and experimental conditions. For example, the Proteintech 14142-1-AP antibody has demonstrated effective Western blot detection at dilutions from 1:1000 to 1:6000, but researchers should titrate the antibody in their specific testing systems to obtain optimal results .

What cell and tissue types have been validated for ISYNA1 antibody detection?

ISYNA1 antibodies have been successfully validated in multiple species and sample types:

For immunohistochemistry applications with human pancreatic cancer tissue, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may also be used as an alternative . When studying ISYNA1 in pancreatic cancer models, researchers should be aware that ISYNA1 expression is lower in pancreatic cancer tissues (39.5%) compared to normal pancreatic tissues (57.9%) .

How should I validate the specificity of my ISYNA1 antibody?

Proper validation of ISYNA1 antibody specificity is essential for reliable research outcomes:

• Molecular weight confirmation: Verify that your antibody detects a protein at the expected molecular weight (approximately 61 kDa for full-length ISYNA1) .

• Positive and negative controls: Include both positive controls (tissues/cells known to express ISYNA1, such as HepG2 cells, mouse/rat testis tissue) and negative controls (samples with ISYNA1 knockdown) .

• siRNA knockdown validation: As demonstrated in multiple studies, siRNA-mediated knockdown of ISYNA1 can confirm antibody specificity. Researchers have successfully used this approach to verify antibody specificity in pancreatic cancer cell lines (Capan-2 and SW1990) .

• Cross-reactivity testing: Test the antibody against samples from different species if conducting comparative studies. Many ISYNA1 antibodies show reactivity with human, mouse, and rat samples .

How can I effectively study the subcellular localization of ISYNA1?

ISYNA1 displays both nuclear and cytoplasmic localization, which can be influenced by experimental conditions and cell types:

• Immunofluorescence protocol for ISYNA1 localization:

  • Fix cells with 4% paraformaldehyde for 1 hour at room temperature

  • Permeabilize with 0.1% Triton X-100 for 5 minutes

  • Block with PBS containing 3% BSA, 1% fish gelatin, 50 mM NH₄Cl, and 5% goat serum overnight at 4°C

  • Incubate with primary ISYNA1 antibody at appropriate dilution (1:20-1:200) for 1 hour at room temperature

  • Wash three times with 1% BSA in PBS (pH 8.0)

  • Incubate with Alexa Fluor 488- or Alexa Fluor 546-conjugated secondary antibodies (1:1000) for 1 hour

Research has demonstrated that ISYNA1 is expressed in both the nucleus and cytoplasm of pancreatic cancer cell lines . When conducting immunofluorescence studies with HepG2 cells, researchers can effectively visualize ISYNA1 using antibody dilutions between 1:20 and 1:200 .

What approaches can I use to study p53-mediated regulation of ISYNA1?

p53 has been identified as a key regulator of ISYNA1 expression, making this an important area for investigation:

• p53 response element (RE) analysis: Two p53 response elements have been identified in the ISYNA1 gene - one in the promoter region (RE1) and another in the seventh exon (RE2). Luciferase reporter assays with these elements can be used to study p53-mediated transcriptional regulation of ISYNA1 .

• Chromatin immunoprecipitation (ChIP): Studies have demonstrated that p53 directly binds to the genomic fragment that includes RE2 in the seventh exon of ISYNA1. ChIP assays can be performed to assess p53 binding to these regions under different experimental conditions .

• p53 knockdown and overexpression:

  • siRNA-mediated silencing of p53 inhibits the induction of ISYNA1 in p53 wild-type cells

  • Wild-type p53 overexpression using adenoviral vectors (Ad-p53) induces ISYNA1 expression

  • Importantly, wild-type p53 (wtp53) positively regulates ISYNA1, while mutant p53 (mtp53) does not show this regulatory effect

A key methodological consideration: when studying the p53-ISYNA1 relationship, researchers should consider the p53 status (wild-type vs. mutant) of their experimental cell lines. For example, Capan-2 and SW1990 cells express wild-type p53, while Miapaca-2 cells express mutant p53 .

How can I investigate the role of ISYNA1 in cell proliferation and tumor progression?

ISYNA1 has been identified as a potential tumor suppressor, particularly in pancreatic cancer:

• ISYNA1 silencing experiments: siRNA-mediated knockdown of ISYNA1 has been demonstrated to:

  • Promote cell proliferation and cell cycle progression by inhibiting p21

  • Enhance cell migration and invasion by upregulating ZEB-1

  • Counteract the anti-tumor effects of MSI2 (Musashi2) silencing

• Colony formation assays: Overexpression of ISYNA1 suppresses cell proliferation in HCT116 and HepG2 cells, while ISYNA1 silencing increases resistance to adriamycin (ADR) treatment .

• Flow cytometry for cell cycle analysis: ISYNA1 silencing decreases the proportion of cells in G1 phase in pancreatic cancer cell lines, indicating an effect on cell cycle regulation .

• Combined analysis with other biomarkers: High MSI2 expression combined with low ISYNA1 expression correlates with worse prognosis in pancreatic cancer patients, suggesting the value of ISYNA1 as part of a prognostic biomarker panel .

What are the optimal storage conditions for ISYNA1 antibodies?

Proper storage is essential for maintaining antibody performance:

• Storage temperature: Store ISYNA1 antibodies at -20°C. They remain stable for one year after shipment when properly stored .

• Buffer composition: Most commercial ISYNA1 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Aliquoting considerations: Aliquoting is generally unnecessary for -20°C storage, but may be advisable for antibodies that will be used repeatedly to avoid freeze-thaw cycles .

• Special formulations: Some commercially available ISYNA1 antibodies (20μl sizes) contain 0.1% BSA, which should be considered when designing experiments that may be sensitive to BSA .

How can I optimize antigen retrieval for ISYNA1 immunohistochemistry?

Effective antigen retrieval is critical for successful ISYNA1 immunohistochemistry:

• Primary recommendation: Use TE buffer at pH 9.0 for antigen retrieval of ISYNA1 in formalin-fixed, paraffin-embedded tissues .

• Alternative method: Citrate buffer at pH 6.0 may also be used as an alternative for antigen retrieval, although this may result in different staining intensity compared to TE buffer .

• Tissue-specific considerations: For pancreatic cancer tissues, the TE buffer method has been validated in multiple studies and appears to provide optimal results for ISYNA1 detection .

• Validation approach: When implementing a new antigen retrieval protocol, compare the results with established positive controls, such as human pancreatic cancer tissue known to express ISYNA1 .

What are potential confounding factors when studying ISYNA1 expression?

Several factors may influence ISYNA1 expression and detection:

• Tissue-specific isoform expression: ISYNA1 displays tissue-specific isoform expression patterns. The brain and testis predominantly express the 68-kDa isoform, while the pancreas has a slightly smaller 67-kDa isoform. The intestine exhibits a unique profile with 62, 43, and 20 kDa isoforms .

• p53 status influence: The p53 status of cell lines or tissues significantly affects ISYNA1 expression. Wild-type p53 positively regulates ISYNA1, while this regulation is absent in cells with mutant p53 .

• Drug treatment effects: Mood-stabilizing drugs like valproate and lithium inhibit ISYNA1, which may affect experimental results if such compounds are present in experimental systems .

• Subcellular localization variability: ISYNA1 can be detected in both the nucleus and cytoplasm, and its distribution may vary depending on cellular conditions and experimental treatments .

How can I use ISYNA1 antibodies to study the connection between inositol metabolism and disease?

ISYNA1's role in inositol metabolism makes it relevant to several disease contexts:

• Cancer research applications:

  • Compare ISYNA1 expression between tumor and normal tissues using immunohistochemistry

  • Correlate ISYNA1 expression with clinical parameters (T stage, vascular permeation) and patient outcomes

  • Study the ISYNA1-p21/ZEB-1 pathway in cancer progression models

• Neuropsychiatric disorder connections:

  • Investigate the effects of mood-stabilizing drugs (valproate, lithium) on ISYNA1 expression and activity

  • Explore the relationship between inositol metabolism and mood regulation

• Metabolic pathway analysis:

  • Use ISYNA1 antibodies to study the rate-limiting step in inositol biosynthesis

  • Investigate the conversion of glucose 6-phosphate to myo-inositol 3-phosphate

What are the most effective approaches for multiplexing ISYNA1 with other biomarkers?

For complex experimental designs requiring multiple markers:

• Dual immunofluorescence protocols:

  • ISYNA1 antibodies can be combined with antibodies against related proteins like p53, p21, and ZEB-1

  • Recommended protocol: Fix cells with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100, block overnight, and use fluorophore-conjugated secondary antibodies with distinct emission spectra

• Sequential immunohistochemistry:

  • For tissue sections, consider sequential staining with ISYNA1 and related markers (MSI2, p53)

  • Serial sections can be used to compare expression patterns of different markers

• Co-localization studies:

  • ISYNA1 can be co-stained with subcellular markers to study its localization

  • Successfully implemented in studies visualizing ISYNA1 in both nuclear and cytoplasmic compartments

How can I quantitatively analyze ISYNA1 expression in research samples?

For rigorous quantitative analysis:

• Western blot quantification:

  • Use β-actin or other housekeeping proteins as loading controls for normalization

  • Employ densitometry software to quantify band intensity ratios

  • Compare expression across different experimental conditions (e.g., with/without p53 knockdown)

• Immunohistochemistry scoring systems:

  • Implement validated scoring systems based on staining intensity and percentage of positive cells

  • In pancreatic cancer studies, ISYNA1 expression has been successfully quantified using scoring systems that combine staining intensity and distribution

• qRT-PCR analysis:

  • Design specific primers for ISYNA1 mRNA quantification

  • Normalize to appropriate reference genes (often GAPDH or β-actin)

  • Compare relative expression levels between experimental conditions

What are emerging applications for ISYNA1 antibodies in preclinical research?

Based on current research trends:

• Therapeutic target validation:

  • ISYNA1 has been identified as a potential therapeutic target in pancreatic cancer, with p53-ISYNA1 pathway showing promise for intervention .

  • Antibodies can be used to validate the efficacy of compounds targeting this pathway.

• Biomarker development:

  • Combined analysis of ISYNA1 with other markers (e.g., MSI2) shows potential for improved prognostic assessment .

  • Antibody-based tissue microarray studies could help validate ISYNA1 as a clinical biomarker.

• Inositol metabolism regulation:

  • ISYNA1 antibodies can help elucidate alternative pathways for inositol polyphosphate production, as suggested by recent research .

  • This could lead to new understandings of cellular signaling and metabolism.

How can researchers address conflicting data on ISYNA1 function and regulation?

When faced with contradictory findings: