NOP53 Antibody, Biotin conjugated

What is NOP53 and why is it an important research target?

NOP53 (also known as PICT-1, PICT1, GLTSCR2, or p60) is a nucleolar protein functioning as a ribosome biogenesis factor. In humans, it consists of 478 amino acid residues with a molecular weight of approximately 54.4 kDa. NOP53 plays a crucial role in the integration of the 5S ribonucleoprotein particle (RNP) into the ribosomal large subunit during ribosome biogenesis . This protein has gained research interest due to its structural role in stabilizing critical interactions during ribosome assembly and its involvement in recruiting the RNA exosome for internal transcribed spacer 2 (ITS2) processing .

What is the subcellular localization of NOP53 and in which tissues is it predominantly expressed?

NOP53 is primarily localized in the nucleus, specifically within the nucleolus. Expression analysis reveals tissue-specific patterns with high expression levels in the heart and pancreas, moderate expression in the placenta, liver, skeletal muscle, and kidney, and comparatively low expression in the brain and lung . When designing experiments targeting NOP53, researchers should consider these expression patterns, particularly when selecting appropriate cell lines or tissue samples for investigation.

What are the advantages of using biotin-conjugated NOP53 antibodies versus unconjugated versions?

Biotin-conjugated NOP53 antibodies offer several methodological advantages over unconjugated versions. The biotin-avidin/streptavidin system provides one of the strongest non-covalent biological interactions known, offering enhanced sensitivity through signal amplification. This allows for:

• Improved detection limits in assays targeting low-abundance NOP53

• Greater flexibility in experimental design through multiple secondary detection options

• Compatibility with various detection systems that utilize streptavidin-conjugated reporters (HRP, fluorophores)

• Reduced background in multi-step detection protocols

• Stability during long-term storage compared to directly labeled antibodies

What applications are biotin-conjugated NOP53 antibodies most suitable for?

• Immunohistochemistry with avidin-biotin complex (ABC) detection

• Flow cytometry with streptavidin-conjugated fluorophores

• Immunoprecipitation coupled with mass spectrometry

• Chromatin immunoprecipitation (ChIP) assays if NOP53 has DNA-associated functions

• Protein microarrays requiring high sensitivity

The choice of application should be guided by the specific research question and experimental design considerations.

How can I validate the specificity of NOP53 antibodies in the context of studying ribosome biogenesis?

Validating NOP53 antibody specificity in ribosome biogenesis studies requires a multi-faceted approach:

• Genetic controls: Compare antibody signals between wild-type cells and those with NOP53 knockdown/knockout. The complete disappearance of signal in knockout samples provides strong validation evidence.

• Subcellular localization confirmation: Since NOP53 is primarily nucleolar, perform co-localization studies with established nucleolar markers (e.g., fibrillarin, nucleolin). Properly functioning NOP53 antibodies should show strong nucleolar enrichment pattern consistent with its role in ribosome assembly .

• Molecular weight verification: In Western blot applications, verify that the detected band corresponds to the expected 54.4 kDa size of NOP53. Multiple bands might indicate either non-specific binding, post-translational modifications, or degradation products.

• Cross-reactivity assessment: If studying NOP53 in non-human systems, verify cross-reactivity with the orthologous protein. NOP53 orthologs have been identified in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken species .

• Comparison with alternative NOP53 antibodies: Using different antibodies recognizing distinct epitopes of NOP53 can further confirm specificity.

What experimental considerations should be taken into account when studying NOP53's role in the transition from nucleolar to nucleoplasmic pre-60S ribosomal particles?

Research investigating NOP53's role in pre-60S ribosomal particle transition from nucleolus to nucleoplasm requires careful experimental design:

• Temporal resolution: As NOP53 replaces Erb1 at a specific stage of ribosome assembly, time-course experiments are essential to capture this dynamic transition .

• Compartment isolation: Employ nucleolar, nucleoplasmic, and cytoplasmic fractionation to track pre-60S particles across cellular compartments. Density gradient centrifugation can separate different ribosomal assembly intermediates.

• Interactome analysis: As NOP53 interacts with multiple proteins (Nop7, Rlp7, eL8, uL29, and uL23), use co-immunoprecipitation with biotin-conjugated NOP53 antibodies followed by mass spectrometry to identify stage-specific interaction partners .

• Structural considerations: The tetrahedral architecture of NOP53 at the basis of the pre-60S foot suggests it may act as a scaffold protein. Experiments should be designed to distinguish between its structural role and its role in recruiting the RNA exosome .

• Mutational studies: Compare wild-type NOP53 with mutants lacking the exosome-interacting motif to differentiate between its exosome recruitment function and structural roles .

How can NOP53 antibodies be optimally used to investigate the differential effects of NOP53 depletion versus truncation on 60S ribosomal subunit assembly?

To investigate differential effects between NOP53 depletion and truncation on 60S ribosomal subunit assembly:

• Establish comparative systems: Generate both NOP53-depleted cells (using siRNA/shRNA) and cells expressing truncated NOP53 mutants (particularly those lacking the arch-interacting motif required for exosome interaction).

• Pre-ribosomal particle isolation: Isolate pre-60S particles from both conditions using established tandem affinity purification protocols with tagged assembly factors like Nog2.

• Quantitative proteomics: Employ MS/MS-based quantitative proteomics to analyze the compositional differences of pre-60S particles between the two conditions. This approach can reveal differential retention or loss of specific assembly factors .

• rRNA processing analysis: Northern blotting and primer extension assays to monitor rRNA intermediates (particularly 7S pre-rRNA) accumulation patterns, which will differ between depletion and truncation scenarios .

• Electron microscopy: Structural analysis using negative staining or cryo-EM to visualize morphological differences in pre-60S particles between the two conditions, focusing on foot structure integrity.

• Functional readouts: Monitor downstream events such as Yvh1 recruitment, which has been shown to be impaired specifically in NOP53 depletion conditions .

What are the optimal conditions for using biotin-conjugated NOP53 antibodies in immunofluorescence to visualize nucleolar dynamics?

For optimal immunofluorescence results with biotin-conjugated NOP53 antibodies:

• Fixation protocol:

  • 4% paraformaldehyde (10 minutes at room temperature) preserves nuclear morphology

  • Methanol fixation (-20°C for 10 minutes) can provide better access to nucleolar antigens

  • Avoid overfixation which can mask epitopes and increase background

• Permeabilization optimization:

  • Use 0.1-0.5% Triton X-100 (10 minutes) for nuclear penetration

  • For dual detection of cytoplasmic and nucleolar proteins, titrate permeabilization conditions

• Blocking parameters:

  • 5% BSA or 10% normal serum (1 hour at room temperature)

  • Include 0.1% Tween-20 to reduce non-specific binding of the biotin-conjugate

• Detection strategy:

  • Use fluorophore-conjugated streptavidin (e.g., Alexa Fluor 488 or 594)

  • Signal amplification can be achieved using the TSA (Tyramide Signal Amplification) system

  • Optimal antibody dilution should be empirically determined (typical starting range: 1:100-1:500)

• Controls and counterstaining:

  • Include a nucleolar marker (e.g., fibrillarin) for co-localization

  • DAPI or Hoechst for nuclear counterstain

  • Biotin blocking kit should be used to minimize endogenous biotin signal

What protocol modifications are necessary when using NOP53 antibodies in pre-ribosomal particle isolation experiments?

When isolating pre-ribosomal particles using NOP53 antibodies:

• Lysis buffer composition:

  • 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl₂

  • 0.1% NP-40 (not stronger detergents that could disrupt nucleolar structures)

  • Protease inhibitors (complete cocktail) and phosphatase inhibitors

  • RNase inhibitors (40 U/mL) to preserve RNA-protein interactions

• Pre-clearing strategy:

  • Pre-clear lysates with streptavidin beads alone to remove endogenous biotinylated proteins

  • Include yeast tRNA (100 μg/mL) to reduce non-specific RNA binding

• Immunoprecipitation approach:

  • Sequential binding: Incubate lysate with biotin-conjugated NOP53 antibodies (4°C, 2-4 hours) followed by streptavidin beads

  • Wash conditions: Minimum 5 washes with decreasing salt concentrations (final wash in buffer without detergent)

• Elution methods:

  • For protein analysis: Boiling in SDS sample buffer

  • For intact RNP analysis: Competitive elution with biotin (2 mM)

  • For structural studies: TEV protease cleavage if using a tandem affinity approach

• Downstream analysis adjustments:

  • For RNA analysis: Phenol-chloroform extraction and ethanol precipitation

  • For protein composition: TCA precipitation before MS sample preparation

  • For EM analysis: Mild glutaraldehyde fixation (0.1%) to stabilize complexes

How can I optimize Western blot protocols specifically for NOP53 detection using biotin-conjugated antibodies?

For optimized Western blot detection of NOP53 using biotin-conjugated antibodies:

• Sample preparation:

  • Nuclear extracts yield better results than whole cell lysates

  • Use NE-PER Nuclear and Cytoplasmic Extraction Reagents or similar kits

  • Load 20-50 μg of nuclear extract protein per lane

• Gel electrophoresis parameters:

  • 10% acrylamide gels provide optimal resolution around 54.4 kDa

  • Include molecular weight markers spanning 40-70 kDa range

  • Run at lower voltage (80-100V) for better resolution

• Transfer conditions:

  • Wet transfer at 30V overnight at 4°C for complete transfer of nuclear proteins

  • PVDF membranes (0.45 μm) perform better than nitrocellulose for nuclear proteins

  • Verify transfer efficiency with reversible Ponceau S staining

• Blocking optimizations:

  • Critical: Use casein-based blockers instead of BSA to avoid endogenous biotin

  • Commercial biotin blocking kits should be used prior to antibody incubation

  • 5% non-fat dry milk in TBST is an economical alternative

• Detection strategy:

  • Streptavidin-HRP (1:5000-1:10000) provides optimal signal-to-noise ratio

  • Enhanced chemiluminescence with extended exposure times (5-10 minutes)

  • For multiplexing, use fluorescent streptavidin conjugates and appropriate imaging systems

• Controls:

  • Include NOP53-depleted samples as negative controls

  • Use β-actin or HDAC1 as loading controls for normalization

What are the potential causes of high background when using biotin-conjugated NOP53 antibodies, and how can these issues be resolved?

High background with biotin-conjugated antibodies typically stems from specific causes requiring targeted solutions:

• Endogenous biotin interference:

  • Problem: Cells naturally contain biotin-containing proteins

  • Solution: Use commercial biotin/avidin blocking kits before applying biotin-conjugated antibodies

  • Alternative: Switch to casein-based blockers instead of BSA (which contains biotin)

• Non-specific binding:

  • Problem: Secondary streptavidin reagents binding to off-target molecules

  • Solution: Add 0.1-0.2% Tween-20 to washing buffers and increase wash frequency (5-6 times)

  • Alternative: Pre-adsorb antibodies with cell/tissue extracts from species distinct from target

• Antibody concentration issues:

  • Problem: Excessive antibody concentration leading to non-specific binding

  • Solution: Titrate antibody concentrations (typical working range: 1:200-1:1000)

  • Alternative: Reduce incubation time (1-2 hours at room temperature rather than overnight)

• Buffer composition problems:

  • Problem: Incompatible buffer components causing precipitation

  • Solution: Ensure all buffers are filtered (0.22 μm) and prepared fresh

  • Alternative: Add 0.1% carrier protein (IgG-free BSA) to stabilize antibody

• Fixation artifacts:

  • Problem: Over-fixation causing autofluorescence or epitope masking

  • Solution: Optimize fixation time and concentration for paraformaldehyde (2-4%, 10 minutes)

  • Alternative: Try acetone fixation for 10 minutes at -20°C

How can I address discrepancies between expected and observed molecular weights when detecting NOP53 with biotin-conjugated antibodies?

Molecular weight discrepancies in NOP53 detection could indicate several biological or technical issues:

• Post-translational modifications:

  • Observation: Higher molecular weight than expected 54.4 kDa

  • Explanation: NOP53 undergoes phosphorylation , potentially altering migration

  • Verification: Treat samples with phosphatase before SDS-PAGE

• Protein degradation:

  • Observation: Lower molecular weight bands or smears

  • Explanation: Proteolytic cleavage during sample preparation

  • Solution: Use fresh protease inhibitor cocktails and keep samples cold throughout

• Alternative splicing/isoforms:

  • Observation: Consistent additional bands across multiple experiments

  • Explanation: Tissue-specific NOP53 variants

  • Verification: Compare with RT-PCR analysis of NOP53 transcripts

• Cross-reactivity:

  • Observation: Unexpected bands that don't respond to NOP53 manipulation

  • Explanation: Antibody binding to related proteins

  • Solution: Validate with additional NOP53 antibodies targeting different epitopes

• Technical artifacts:

  • Observation: Distorted migration patterns

  • Explanation: Salt concentration or sample heating issues

  • Solution: Normalize salt concentration across samples and ensure complete denaturation

What strategies can overcome limited detection sensitivity when using NOP53 antibodies in tissues with low expression levels?

For improved detection in tissues with low NOP53 expression (e.g., brain and lung ):

• Signal amplification techniques:

  • Tyramide Signal Amplification (TSA): Can increase sensitivity 10-100 fold

  • Streptavidin-biotin chain approach: Apply streptavidin followed by biotinylated enzyme

  • Polymer-based detection systems: Use anti-biotin antibody coupled to HRP-polymer

• Sample enrichment approaches:

  • Nuclear fractionation to concentrate NOP53-containing compartments

  • Immunoprecipitation before detection to concentrate target protein

  • Ultracentrifugation to isolate nucleoli specifically

• Microscopy enhancements:

  • Confocal microscopy with increased laser power and detector gain

  • Deconvolution techniques to improve signal-to-noise ratio

  • Super-resolution microscopy (STED, STORM) for detailed nucleolar localization

• Molecular enhancement strategies:

  • Consensus epitope targeting: Select antibodies against highly conserved regions

  • Dual antibody approach: Use two different NOP53 antibodies simultaneously

  • Proximity ligation assay (PLA) to detect NOP53 and known interaction partners

• Detection system optimization:

  • Enhanced chemiluminescence-plus (ECL+) substrates for Western blots

  • Extended enzyme substrate incubation times for colorimetric detection

  • Fluorescent streptavidin conjugates with high quantum yield fluorophores