NUSAP1 Antibody

What applications are NUSAP1 antibodies validated for?

NUSAP1 antibodies have been validated for multiple research applications:

For optimal results, antibody dilutions should be titrated for each specific experimental system .

How should NUSAP1 antibodies be stored and handled?

NUSAP1 antibodies are typically supplied in PBS buffer with 0.02% sodium azide and 50% glycerol at pH 7.3. They should be stored at -20°C where they remain stable for up to one year after shipment. Aliquoting is unnecessary for -20°C storage. Some preparations may contain 0.1% BSA for increased stability. Always follow manufacturer specifications for the specific antibody being used .

What is the expected molecular weight band for NUSAP1 in Western blotting?

The calculated molecular weight of NUSAP1 is 49 kDa (440 amino acids), but the observed molecular weight typically appears between 47-52 kDa on SDS-PAGE gels. This slight discrepancy may be due to post-translational modifications or the specific sample preparation methods .

What controls should be included when using NUSAP1 antibodies?

For rigorous experimental design when using NUSAP1 antibodies:

• Positive controls: Use cell lines known to express NUSAP1 such as HEK-293, HeLa, or Jurkat cells

• Negative controls: Include:

  • Primary antibody omission control

  • NUSAP1 knockdown samples (RNAi depletion has been shown to completely eliminate staining, confirming antibody specificity)

• Loading controls: For Western blots, use housekeeping proteins like GAPDH

• Isotype controls: Include matched isotype rabbit IgG for polyclonal antibodies

These controls are essential for validating specificity and reducing false-positive results .

How can I optimize immunohistochemistry (IHC) protocols for NUSAP1 detection?

For optimal IHC detection of NUSAP1:

• Antigen retrieval method: Use TE buffer at pH 9.0 (primary recommendation) or citrate buffer at pH 6.0 (alternative)

• Blocking procedure: Block with goat serum for 30 minutes at 37°C

• Antibody incubation: Use 1:100 working concentration and incubate overnight at 4°C

• Visualization system: 3,3'-Diaminobenzidine (DAB) system works effectively

• Scoring method: Implement a combined scoring system considering both staining intensity and percentage of stained cells:

  • 0-2 scores (–)

  • 3-4 scores (+)

  • 5-8 scores (++)

  • 9-12 scores (+++)

This protocol has been validated in studies examining NUSAP1 expression in ovarian cancer tissues .

How does NUSAP1 expression change throughout the cell cycle?

NUSAP1 expression is cell cycle-dependent with distinct dynamics:

• Protein levels: Peak during mitosis and decrease rapidly following cell division

• Temporal pattern: Following synchronized release from nocodazole block, NUSAP1 protein levels remain detectable for approximately 28 hours

• Subcellular localization changes:

  • Prometaphase: Diffuse staining pattern without specific structure localization

  • Later mitotic phases: More organized localization, though not uniformly distributed across the entire mitotic spindle like most microtubule-binding proteins

  • Interphase: Minimal expression

For accurate assessment of NUSAP1 cell cycle dynamics, immunofluorescent imaging coupled with synchronized cell populations provides the most reliable results .

What cell types and tissues show significant NUSAP1 expression?

NUSAP1 shows differential expression across tissues and cell types:

• High expression:

  • CD34+CD90+ hematopoietic stem cells (HSCs)

  • Leukemic cell lines

  • B lymphoblasts

  • Various cancer tissues (notably in HCC, ovarian cancer, and AML)

• Low expression:

  • Most differentiated normal tissues

This expression pattern suggests NUSAP1 may serve as a marker for stem-like or proliferative cell populations, particularly in cancer contexts .

How does NUSAP1 expression correlate with cancer prognosis and progression?

NUSAP1 has significant correlations with cancer outcomes:

For prognostic studies, immunohistochemical analysis with standardized scoring systems provides the most clinically relevant data .

Can NUSAP1 antibodies be used to evaluate chemotherapy resistance mechanisms?

NUSAP1 has been implicated in chemotherapy resistance pathways:

• Cisplatin resistance in HCC:

  • NUSAP1 knockdown enhances sensitivity to cisplatin

  • siRNA-mediated NUSAP1 suppression results in:

    • Decreased IC50 values for cisplatin

    • Increased DNA damage (measured by comet assay)

    • Elevated γ-H2AX protein expression (DNA damage marker)

• Experimental approach:

  • Use NUSAP1 antibodies in Western blot to assess NUSAP1 expression

  • Compare control vs. knockdown cells treated with different cisplatin concentrations

  • Combine with DNA damage assays (comet assay and γ-H2AX immunoblotting)

This experimental workflow can effectively evaluate the role of NUSAP1 in chemoresistance mechanisms .

How can NUSAP1 antibodies be used to study tumor immune microenvironment?

NUSAP1 expression correlates with immune cell infiltration in tumors, making it valuable for immune microenvironment studies:

• Correlation analysis approaches:

  • Compare NUSAP1 expression with 22 immune cell subtypes using computational methods

  • Divide samples into high/low NUSAP1 expression groups and analyze differences in immune infiltration

• Significant immune cell correlations (across multiple datasets):

  • T cells CD4 memory resting and activated

  • Macrophages M0 and M2

  • Dendritic cells resting

  • NK cells resting

  • B cells memory

  • T cells regulatory (Tregs)

• Experimental workflow:

  • Use NUSAP1 antibodies for IHC or IF to assess expression in tumor samples

  • Perform multicolor immunofluorescence to co-localize NUSAP1 with immune cell markers

  • Correlate NUSAP1 expression with immune cell quantification

This approach has been validated in HCC studies using both ICGC and TCGA databases .

What is the relationship between NUSAP1 expression and immune checkpoint molecules?

NUSAP1 shows significant positive correlations with immune checkpoint molecules:

• Analysis method:

  • Divide samples into high/low NUSAP1 expression groups

  • Compare expression levels of key immune checkpoint molecules between groups

  • Perform correlation analysis between NUSAP1 and checkpoint molecules

• Key findings:

  • High NUSAP1 expression correlates with elevated levels of immune checkpoint molecules

  • Positive correlations observed with multiple checkpoint molecules

  • Suggests potential role in immunotherapy response prediction

• Application in research:

  • NUSAP1 antibodies can be used to stratify patient samples for immunotherapy response studies

  • May serve as companion biomarker for immune checkpoint inhibitor therapy

These correlations suggest NUSAP1 may be valuable as a predictive biomarker for immunotherapy response .

What are common issues with NUSAP1 antibodies and how can they be resolved?

Common technical challenges and solutions:

• High background in immunostaining:

  • Increase blocking time and concentration (5% BSA or 10% normal serum)

  • Optimize antibody concentration (start with 1:100 dilution for IHC/IF)

  • Ensure proper washing steps (minimum 3x5 minutes with PBST)

• Multiple bands in Western blot:

  • Verify expected molecular weight (47-52 kDa)

  • Increase blocking stringency

  • Use freshly prepared lysates to minimize degradation

  • Include protease inhibitor cocktails in lysis buffers

• Weak or no signal in cell cycle studies:

  • Consider cell synchronization (NUSAP1 expression peaks during mitosis)

  • Verify mitotic index in your samples (NUSAP1 expression is highest in M phase)

  • Use positive control cell lines (HeLa, Jurkat cells)

• Antibody validation approach:

  • Perform siRNA knockdown experiments as validation controls

  • Compare results across multiple antibody clones if available

These troubleshooting approaches are based on published methodologies and technical protocols .

How can I quantify NUSAP1 expression levels across different experimental conditions?

For accurate NUSAP1 quantification:

• Western blot quantification:

  • Use LI-COR or other quantitative blotting systems

  • Normalize to loading controls (GAPDH)

  • Use gradient dilutions to ensure linearity of signal

• IHC scoring systems:

  • Implement standardized scoring system (e.g., H-score = intensity × percentage)

  • Consider digital pathology for more objective quantification

  • Use automated analysis software when possible

• Flow cytometry approach:

  • Suitable for cell cycle studies

  • Combine with cell cycle markers (PI staining)

  • Analyze mean fluorescence intensity (MFI)

• RT-qPCR normalization:

  • Reference genes: GAPDH, β-actin

  • Use Δ-Δ Ct method for relative quantification

  • Validate PCR efficiency with standard curves

These quantification methods have been employed in studies examining NUSAP1 in cancer cells and tissues .

How can NUSAP1 antibodies be used to study DNA damage repair mechanisms?

NUSAP1 has emerging roles in DNA damage response pathways:

• Experimental approaches:

  • Combine NUSAP1 antibodies with DNA damage markers (γ-H2AX, 53BP1)

  • Perform co-immunoprecipitation to identify NUSAP1 interaction partners in DNA repair complexes

  • Assess NUSAP1 localization changes before/after DNA damage induction

• Relevance to cancer research:

  • NUSAP1 knockdown increases cisplatin-induced DNA damage

  • Correlation with BRCA1/2 expression suggests potential role in homologous recombination

  • Associated with p53 signaling pathway

• Recommended methodology:

  • Comet assay to quantify DNA damage

  • Immunofluorescence co-localization studies

  • Proximity ligation assay (PLA) to detect protein-protein interactions

These approaches can elucidate the emerging role of NUSAP1 in DNA damage response and repair .

What is the potential of NUSAP1 as a therapeutic target, and how can antibodies contribute to this research?

NUSAP1 shows promise as a therapeutic target:

• Target validation approaches:

  • Use antibodies to assess NUSAP1 expression across patient samples

  • Correlate expression with treatment outcomes

  • Perform siRNA or CRISPR knockdown to evaluate phenotypic effects

• Therapeutic development applications:

  • Screen for small molecule inhibitors that disrupt NUSAP1 function

  • Use antibodies to evaluate target engagement

  • Develop antibody-drug conjugates targeting NUSAP1

• Biomarker applications:

  • Stratify patients for clinical trials

  • Monitor treatment response

  • Predict prognosis in various cancer types

• Combined approaches:

  • Assess NUSAP1 inhibition in combination with established therapies

  • Evaluate synergy with DNA-damaging agents (e.g., cisplatin)

  • Explore potential with immunotherapeutic approaches

These research directions leverage NUSAP1's roles in cell cycle regulation, cancer progression, and potential impact on therapeutic response .