NOP53 Antibody, FITC conjugated

What is NOP53 protein and why is it significant for research?

NOP53 (also known as GLTSCR2 or PICT-1) functions as a ribosome biogenesis protein predominantly localized in the nucleolus. It plays crucial roles in pre-rRNA processing and ribosome assembly, particularly in the maturation of 60S ribosomal subunits. Recent research has identified NOP53 as an adaptor protein responsible for recruiting the RNA exosome complex during the processing of 7S pre-rRNA to mature 5.8S rRNA . Beyond ribosomal functions, NOP53 has been implicated in regulating the DNA damage response and influencing radiotherapy resistance in cancer cells by suppressing p53 activation . The protein's dual role in fundamental cellular processes and pathological conditions makes it a compelling target for both basic and translational research.

What are the key specifications of NOP53 Antibody, FITC conjugated for research applications?

NOP53 Antibody, FITC conjugated is a polyclonal antibody developed in rabbits using recombinant Human Ribosome biogenesis protein NOP53 (amino acids 227-405) as the immunogen . The antibody specifically targets human NOP53 and has been validated for ELISA applications. Key specifications include:

The FITC conjugation enables direct fluorescence detection without requiring secondary antibodies, making it particularly valuable for techniques requiring multicolor immunofluorescence or flow cytometry .

How should researchers validate NOP53 Antibody, FITC conjugated for their specific experimental systems?

Proper validation of NOP53 Antibody, FITC conjugated requires a systematic approach to ensure specificity and reliability across different experimental systems:

• Positive control validation: Use cell lines with known NOP53 expression (such as colorectal cancer cell lines) for initial validation . Compare staining patterns with established nucleolar markers like NPM1, which has been shown to colocalize with NOP53 .

• Negative control implementation: Include relevant negative controls including:

  • Secondary antibody-only controls (when using the antibody in non-direct detection methods)

  • Isotype controls with FITC-conjugated rabbit IgG

  • Cell lines with NOP53 knockdown/knockout (if available)

• Cross-validation with other detection methods: Verify results using complementary techniques such as Western blotting with non-conjugated NOP53 antibodies or RNA expression analysis.

• Subcellular localization assessment: NOP53 should predominantly localize to the nucleolus and colocalize with nucleolar markers. Immunofluorescence studies have consistently shown NOP53 colocalizing with NPM1 in both cell lines and clinical specimens .

• Specificity testing: Preabsorption of the antibody with recombinant NOP53 protein should abolish or significantly reduce the signal if the antibody is specific.

This comprehensive validation approach ensures that any experimental results obtained using this antibody can be interpreted with confidence and reproducibility.

What are the optimal protocols for immunofluorescence studies using NOP53 Antibody, FITC conjugated?

For optimal immunofluorescence results with NOP53 Antibody, FITC conjugated, researchers should follow this detailed protocol designed to preserve nucleolar architecture and maximize signal-to-noise ratio:

• Cell preparation:

  • Culture cells on poly-L-lysine coated coverslips

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

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

• Blocking and antibody incubation:

  • Block with 3% BSA in PBS for 1 hour at room temperature

  • Incubate with NOP53 Antibody, FITC conjugated (1:100-1:500 dilution, optimized for each lot) in blocking buffer for 2 hours at room temperature or overnight at 4°C

  • Wash 3x with PBS containing 0.1% Tween-20

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes

  • Mount with anti-fade mounting medium

  • For colocalization studies with nucleolar markers, include appropriate antibodies (e.g., anti-NPM1) with compatible fluorophores

• Microscopy considerations:

  • Use confocal microscopy for precise nucleolar localization

  • Employ appropriate filter sets for FITC (excitation ~495 nm, emission ~519 nm)

  • Adjust exposure settings to avoid photobleaching of the FITC fluorophore

This protocol has been effectively applied in studies demonstrating colocalization of NOP53 with nucleolar protein NPM1 in both colorectal cancer cell lines and patient samples .

How can researchers utilize NOP53 Antibody, FITC conjugated to study liquid-liquid phase separation (LLPS) in the nucleolus?

LLPS has emerged as a critical mechanism in nucleolar organization and function. NOP53 has been identified as a protein capable of undergoing LLPS, forming liquid-like condensates in the nucleolus . To investigate LLPS properties of NOP53 using the FITC-conjugated antibody:

• Live cell imaging approach:

  • Transfect cells with fluorescent protein-tagged NOP53 constructs

  • Use the NOP53 Antibody, FITC conjugated to confirm proper localization and behavior of tagged constructs

  • Perform time-lapse imaging to observe fusion events characteristic of liquid droplets

• LLPS disruption assays:

  • Treat cells with 1,6-hexanediol (5-10%) to disrupt weak hydrophobic interactions

  • Track changes in NOP53 condensate morphology and distribution using the antibody

  • Compare results with known LLPS proteins as positive controls

• Fluorescence Recovery After Photobleaching (FRAP):

  • Bleach a small region within NOP53-enriched nucleolar regions

  • Monitor fluorescence recovery rate to assess molecular dynamics

  • Analyze FRAP curves to determine mobile fraction and half-time of recovery

• Domain-specific analysis:

  • Compare wild-type NOP53 versus mutants lacking the intrinsically disordered region 1 (IDR1)

  • IDR1 has been shown to be required for NOP53 condensate formation

  • The antibody can be used to detect changes in localization patterns with different mutants

• Multivalent interactions investigation:

  • Study how multivalent-arginine-rich linear motifs (M-R motifs) affect nucleolar localization

  • These motifs are essential for proper nucleolar targeting but dispensable for LLPS behavior

This methodological approach provides a comprehensive examination of NOP53's LLPS properties and nucleolar dynamics, contributing to our understanding of how biomolecular condensates regulate ribosome biogenesis.

What approaches should be used when studying NOP53's role in the exosome complex recruitment using the FITC-conjugated antibody?

NOP53 functions as an adaptor protein that recruits the RNA exosome complex for processing 7S pre-rRNA to mature 5.8S rRNA . When investigating this function:

• Colocalization analysis with exosome components:

  • Perform dual immunofluorescence with NOP53 Antibody, FITC conjugated and antibodies against exosome components (Rrp6, Rrp43)

  • Quantify colocalization using Pearson's correlation coefficient or Manders' overlap coefficient

  • Focus analysis on pre-60S particles within nucleoli and nucleoplasm

• Functional depletion studies:

  • Use tetracycline-regulated or galactose-regulated systems to deplete NOP53

  • Monitor changes in exosome localization and pre-rRNA processing

  • The experimental design should follow established protocols where cells are shifted from galactose to glucose media for 18 hours, or treated with doxycycline for 18 hours in tetracycline-regulated systems

• Protein-protein interaction validation:

  • Combine immunofluorescence with proximity ligation assays

  • Verify interactions between NOP53 and specific exosome components (Rrp6, Rrp45, Mpp6)

  • Correlate microscopy data with biochemical interaction data from pulldown assays

• Domain-specific interaction mapping:

  • Generate truncation mutants of NOP53 (similar to those described in the literature)

  • Assess how different domains affect exosome recruitment and positioning

  • The antibody can help track localization changes of these mutants if the epitope region is preserved

This systematic approach will help elucidate how NOP53 not only recruits but also correctly positions the exosome complex during 60S ribosomal subunit maturation, expanding our understanding of ribosome assembly dynamics .

How can researchers apply NOP53 Antibody, FITC conjugated to investigate radiotherapy resistance mechanisms in cancer?

Recent studies have established NOP53's role in promoting radiotherapy resistance in colorectal cancer through suppression of the p53 pathway . To investigate this mechanism:

• Radiation response analysis protocol:

  • Expose cancer cells to clinically relevant radiation doses (2-10 Gy)

  • Use NOP53 Antibody, FITC conjugated for immunofluorescence at defined time points (0, 6, 12, 24 hours post-irradiation)

  • Quantify changes in NOP53 expression, localization, and condensate formation

  • Correlate with markers of DNA damage (γH2AX) and p53 activation

• Clinical correlation methodology:

  • Apply the antibody to tissue microarrays from patients with known radiation response outcomes

  • Develop standardized scoring systems for NOP53 expression levels

  • Utilize digital pathology approaches for quantitative assessment

  • This approach has revealed associations between high NOP53 expression and poor response to neoadjuvant chemoradiotherapy

• Mechanistic pathway investigation:

  • Combine NOP53 immunofluorescence with p53 pathway components

  • Analyze temporal dynamics following irradiation

  • Implement NOP53 knockdown/overexpression systems to manipulate radioresistance

  • Correlate LLPS behavior with radioresistance mechanisms

• Therapeutic targeting strategy development:

  • Screen compounds that disrupt NOP53 LLPS or its interaction with p53 pathway components

  • Use the antibody to monitor effects on NOP53 localization and function

  • Evaluate combination approaches with radiotherapy

This research framework leverages the FITC-conjugated antibody to develop comprehensive insights into how NOP53 contributes to radiotherapy resistance, potentially identifying novel sensitization strategies for cancer treatment.

What are the best experimental designs for studying NOP53's interactions with other nucleolar proteins?

To effectively investigate NOP53's protein interaction network within the nucleolus:

• Proximity-based interaction analysis:

  • Implement BioID or APEX proximity labeling with NOP53 as the bait protein

  • Use NOP53 Antibody, FITC conjugated to verify proper localization of fusion constructs

  • Confirm interactions through colocalization studies with candidate partners

• Domain-specific interaction mapping:

  • Generate a series of NOP53 truncation mutants similar to those described in the literature :

    • NOP53(1-80)

    • NOP53(81-157)

    • NOP53(161-230)

    • NOP53(1-300)

    • NOP53(301-380)

    • NOP53(382-455)

  • Use the antibody to confirm expression and localization if the epitope is preserved

  • Perform co-immunoprecipitation or GST-pulldown assays to map interaction domains

• RNA-dependent interaction assessment:

  • Treat samples with RNase before immunoprecipitation

  • Determine which interactions are RNA-dependent versus direct protein-protein interactions

  • Correlate findings with rRNA processing defects

• Competitive binding experiments:

  • Design peptides derived from interaction interfaces

  • Evaluate disruption of specific interactions

  • Use the antibody to track changes in localization or complex formation

• Quantitative interaction dynamics:

  • Implement fluorescence correlation spectroscopy or number and brightness analysis

  • Measure interaction kinetics and stoichiometry

  • Correlate with functional outcomes in ribosome biogenesis

This comprehensive approach will generate a detailed map of NOP53's interaction network, providing insights into its multifunctional roles in nucleolar processes and cancer-related mechanisms.

How can researchers effectively investigate NOP53 expression across different cancer types and correlate with patient outcomes?

For comprehensive cancer-related NOP53 studies:

This methodological framework enables rigorous investigation of NOP53 as a potential biomarker for cancer prognosis and treatment response, particularly in the context of radiotherapy resistance.

What are common issues when using NOP53 Antibody, FITC conjugated and how can they be resolved?

Researchers may encounter several challenges when working with NOP53 Antibody, FITC conjugated. Here are common problems and their solutions:

• Weak or absent signal:

  • Cause: Suboptimal antibody concentration, inadequate permeabilization, or epitope masking

  • Solution: Titrate antibody concentrations (1:50 to 1:500), increase permeabilization time (up to 15 minutes with 0.2% Triton X-100), or implement antigen retrieval (citrate buffer pH 6.0 at 95°C for 15-20 minutes)

• High background fluorescence:

  • Cause: Insufficient blocking, excessive antibody concentration, or non-specific binding

  • Solution: Extend blocking time (2 hours or overnight), reduce antibody concentration, increase washing steps (5x5 minutes), or add 0.1% Tween-20 to washing buffer

• Photobleaching:

  • Cause: FITC is relatively prone to photobleaching

  • Solution: Use anti-fade mounting medium, minimize exposure to light during preparation, reduce excitation intensity, or consider taking images from unexposed areas of the sample

• Nucleolar structural disruption:

  • Cause: Harsh fixation or permeabilization conditions

  • Solution: Use freshly prepared 4% paraformaldehyde, optimize fixation time (10-15 minutes), and adjust permeabilization conditions

• Inconsistent staining patterns:

  • Cause: Cell cycle variations or heterogeneous protein expression

  • Solution: Synchronize cells if appropriate, increase sample size for quantification, and correlate with cell cycle markers

• Nuclear versus nucleolar localization discrepancies:

  • Cause: NOP53 can relocalize under stress conditions

  • Solution: Control experimental conditions carefully, document cell culture conditions, and monitor potential stressors

Implementation of these troubleshooting approaches will improve experimental reliability and data quality when working with NOP53 Antibody, FITC conjugated.

How should researchers optimize protocols for studying NOP53's dynamic behavior during cell cycle progression?

NOP53 exhibits dynamic localization and potentially changing functions throughout the cell cycle. To study these dynamics:

• Cell synchronization protocol optimization:

  • Implement double thymidine block for G1/S boundary arrest

  • Use nocodazole (100 ng/ml, 12-16 hours) for mitotic arrest

  • Perform serum starvation (0.1% serum, 48 hours) for G0/G1 arrest

  • Release cells and collect at defined timepoints (0, 2, 4, 6, 8, 10, 12 hours)

• Multiparameter analysis setup:

  • Combine NOP53 Antibody, FITC conjugated with cell cycle markers:

    • Cyclin D1 (G1)

    • PCNA or EdU incorporation (S phase)

    • Phospho-Histone H3 (M phase)

  • Use compatible fluorophores for multiplexed imaging

  • Apply quantitative image analysis to measure changes in:

    • NOP53 signal intensity

    • Nucleolar versus nucleoplasmic distribution

    • Colocalization with other nucleolar proteins

• Live cell imaging adaptation:

  • Transfect cells with fluorescent protein-tagged NOP53

  • Validate localization patterns match antibody staining

  • Perform time-lapse microscopy through cell cycle progression

  • Correlate with FUCCI cell cycle reporters

• Nucleolar stress response integration:

  • Combine cell cycle analysis with nucleolar stress inducers

  • Monitor changes in NOP53 localization and LLPS behavior

  • Correlate with ribosome biogenesis markers

This comprehensive approach will provide insights into how NOP53's function, localization, and interaction network may be regulated throughout the cell cycle, with implications for both normal cellular physiology and cancer pathobiology.

How can NOP53 Antibody, FITC conjugated be utilized to investigate the relationship between ribosome biogenesis and DNA damage response?

Recent research has established connections between ribosome biogenesis and DNA damage response pathways, with NOP53 potentially serving as a molecular link . To investigate this relationship:

• Integrated stress response analysis:

  • Expose cells to different stressors:

    • Radiation (2-10 Gy)

    • Nucleolar stress (low-dose actinomycin D, 5-10 nM)

    • Replication stress (hydroxyurea)

  • Track NOP53 localization and condensate formation using the antibody

  • Correlate with markers of:

    • Nucleolar stress (NPM1 translocation)

    • DNA damage (γH2AX foci)

    • p53 activation (phospho-p53, p21 induction)

• Domain-specific function dissection:

  • Generate NOP53 constructs with mutations in:

    • IDR1 (required for LLPS)

    • M-R motifs (required for nucleolar localization)

    • Interaction domains for exosome components

  • Assess how these mutations affect both ribosome biogenesis and DNA damage response

  • Use the antibody to detect changes in localization and interaction patterns

• Temporal dynamics investigation:

  • Implement precise time-course studies following DNA damage

  • Document the sequence of events involving NOP53 relocalization

  • Determine whether NOP53 acts as a sensor, mediator, or effector in connecting these processes

• Therapeutic targeting opportunities:

  • Screen compounds that selectively disrupt NOP53's role in radioresistance

  • Identify molecules that affect LLPS behavior without impairing ribosome biogenesis

  • Use the antibody to monitor drug effects on NOP53 localization and function

This research direction could reveal novel therapeutic vulnerabilities in cancer cells with dysregulated ribosome biogenesis and DNA damage response pathways.

What methodological approaches can researchers use to study NOP53's role in colorectal cancer using the FITC-conjugated antibody?

Building on findings linking NOP53 to colorectal cancer radioresistance , researchers can implement these methodological approaches:

• Patient-derived organoid models:

  • Establish organoids from colorectal cancer patients with varying radiation responses

  • Implement immunofluorescence protocols optimized for 3D structures

  • Correlate NOP53 expression patterns with radiation sensitivity

  • Perform gene editing to modulate NOP53 expression or function

• In situ analysis of clinical specimens:

  • Apply multiplex immunofluorescence to tissue microarrays

  • Include markers for:

    • Proliferation (Ki-67)

    • Stemness (LGR5, CD44)

    • DNA damage response (γH2AX, 53BP1)

    • p53 pathway components

  • Develop tissue-specific quantification algorithms

• Mechanistic pathway investigation:

  • Implement genetic screens (CRISPR, RNAi) to identify synthetic lethal interactions

  • Focus on genes that, when inhibited, sensitize NOP53-high cells to radiation

  • Use the antibody to confirm effective NOP53 targeting

• Translational model development:

  • Establish patient-derived xenograft models from NOP53-high tumors

  • Test combination therapies targeting NOP53-dependent pathways

  • Develop companion diagnostic approaches based on NOP53 expression

This comprehensive approach leverages the FITC-conjugated NOP53 antibody to advance both basic understanding of colorectal cancer biology and development of targeted therapeutic strategies to overcome radioresistance.

How can researchers apply cutting-edge imaging techniques with NOP53 Antibody, FITC conjugated to study nucleolar organization?

Emerging super-resolution and quantitative imaging approaches offer new opportunities to study NOP53's role in nucleolar organization:

• Super-resolution microscopy implementation:

  • Apply structured illumination microscopy (SIM) to achieve 120 nm resolution

  • Implement stimulated emission depletion (STED) microscopy for even higher resolution

  • Use stochastic optical reconstruction microscopy (STORM) to achieve molecular-scale resolution

  • These techniques can reveal precise localization of NOP53 within nucleolar subcompartments (fibrillar center, dense fibrillar component, granular component)

• Expansion microscopy adaptation:

  • Protocol modification for nucleolar proteins:

    • Optimize fixation to preserve nucleolar structure

    • Adjust digestion conditions to maintain epitope accessibility

    • Implement post-expansion staining if needed

  • Combine with conventional confocal microscopy for improved resolution

  • Correlate NOP53 localization with rRNA processing sites

• Live-cell dynamics quantification:

  • Combine with fluorescence recovery after photobleaching (FRAP)

  • Implement fluorescence correlation spectroscopy (FCS)

  • Apply raster image correlation spectroscopy (RICS)

  • These techniques can quantify diffusion rates, binding kinetics, and molecular interactions

• Correlative light and electron microscopy (CLEM):

  • Localize NOP53 using the FITC-conjugated antibody

  • Process the same sample for electron microscopy

  • Correlate fluorescence signal with ultrastructural features

  • This approach bridges molecular specificity with ultrastructural context

These advanced imaging approaches will provide unprecedented insights into how NOP53 contributes to nucleolar organization and function, particularly in the context of liquid-liquid phase separation and ribosome biogenesis.