UTP18 Antibody, HRP conjugated

What is UTP18 and what cellular functions does it perform?

UTP18, also known as WD Repeat Domain 50 (WDR50), is a critical component of the small subunit (SSU) processome, which serves as the first precursor of the small eukaryotic ribosomal subunit. This protein plays a vital role in ribosome biogenesis, specifically in the nucleolar processing of pre-18S ribosomal RNA .

During the assembly of the SSU processome in the nucleolus, UTP18 works alongside numerous ribosome biogenesis factors, RNA chaperones, and ribosomal proteins. These factors collectively associate with nascent pre-rRNA to facilitate essential processes including RNA folding, modifications, structural rearrangements, cleavage, and targeted degradation of pre-ribosomal RNA by the RNA exosome .

UTP18 forms part of the UTP B subcomplex, a crucial building block required for proper assembly and function of the SSU processome. In yeast models, UTP18 forms a stable trimeric structure with Utp6 and Utp21, which is essential for the formation of the tetrameric core complex of the UTP B subcomplex . This hierarchical assembly is critical for proper ribosome biogenesis and, consequently, cellular protein synthesis capacity.

What are the different types of UTP18 antibodies available and what are their specifications?

Multiple UTP18 antibodies are available with varying specifications to accommodate different experimental needs:

The HRP-conjugated UTP18 antibody specifically targets amino acids 13-152 of the human UTP18 protein and is particularly suitable for ELISA applications . This antibody has been generated in rabbits as a polyclonal reagent, providing broad epitope recognition. The conjugation to Horseradish Peroxidase (HRP) enables direct detection without the need for secondary antibodies, streamlining experimental workflows and potentially reducing background signals.

What are the recommended applications for UTP18 antibody, HRP conjugated?

The HRP-conjugated UTP18 antibody is primarily recommended for Enzyme-Linked Immunosorbent Assay (ELISA) applications . Researchers should consider the following methodological guidelines when working with this antibody:

ELISA Applications:

• Direct ELISA: Coat the plate with your sample containing UTP18, then detect directly with the HRP-conjugated antibody. This eliminates the need for a secondary antibody step.

• Sandwich ELISA: Use a capture antibody targeting a different epitope of UTP18, then detect bound UTP18 with the HRP-conjugated antibody.

• Competitive ELISA: Pre-incubate the HRP-conjugated antibody with free UTP18 antigen before adding to the plate coated with UTP18 or anti-UTP18 antibody.

Potential Western Blot Applications:
While the HRP-conjugated version is primarily optimized for ELISA, unconjugated versions of the UTP18 antibody have been validated for Western blot applications . Based on data from unconjugated UTP18 antibodies, the following Western blot conditions may serve as a starting point:

• Antibody concentration: 0.04-0.1 μg/mL

• Sample amount: 5-50 μg of whole cell lysate

• Detection method: Enhanced chemiluminescence (ECL)

• Expected band size: 62 kDa

Immunoprecipitation Considerations:
For researchers interested in protein interaction studies, some UTP18 antibodies have been validated for immunoprecipitation . When performing IP:

• Use NETN lysis buffer for sample preparation

• Antibody amount: approximately 3 μg per mg of lysate

• Loading 20% of immunoprecipitate for subsequent detection

How should UTP18 antibody, HRP conjugated be stored and handled for optimal performance?

Proper storage and handling of HRP-conjugated UTP18 antibody is critical for maintaining its functionality and specificity. Based on standard protocols for HRP-conjugated antibodies and the information available for UTP18 antibodies, the following guidelines are recommended:

Storage Conditions:

• Store at -20°C for long-term storage

• For frequently used antibodies, aliquot and store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles to prevent loss of activity

• Store in buffered aqueous glycerol solution to maintain stability

Handling Recommendations:

• Thawing Process: Allow the antibody to thaw completely at 4°C before use

• Temperature Sensitivity: Keep the antibody on ice during experimental procedures

• Light Sensitivity: HRP conjugates may be sensitive to strong light; store in amber tubes or wrapped in foil

• Working Dilutions: Prepare working dilutions immediately before use and discard any unused diluted antibody

• Contamination Prevention: Use sterile techniques when handling the antibody to prevent microbial contamination

Stability Considerations:

• HRP activity can be affected by strong oxidizing and reducing agents

• Avoid buffers containing sodium azide as they can inhibit HRP enzymatic activity

• Antibody may be stable for up to 12 months when stored properly, but validate activity before critical experiments if stored for extended periods

Quality Control Checks:
Before using in critical experiments, consider performing a simple dot blot or ELISA with positive controls to confirm that the HRP conjugate is still active, especially if the antibody has been stored for extended periods.

Advanced Research Questions

Detecting UTP18 in different cellular compartments requires optimized protocols based on the subcellular localization of this protein and the properties of HRP-conjugated antibodies. Since UTP18 is primarily involved in nucleolar processing of pre-18S ribosomal RNA as part of the SSU processome , its detection often focuses on nuclear and nucleolar compartments.

Subcellular Fractionation Protocol for UTP18 Detection:

• Nucleolar Fraction Isolation:

  • Harvest cells in exponential growth phase

  • Wash cells in PBS and resuspend in buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT, protease inhibitors)

  • Swell cells on ice for 10 minutes and lyse using a Dounce homogenizer

  • Centrifuge at 200× g for 5 minutes to collect nuclei

  • Sonicate nuclei and layer over sucrose cushion

  • Centrifuge at 3000× g for 10 minutes to pellet nucleoli

  • Verify enrichment using nucleolar markers (fibrillarin or nucleolin)

• Optimization of Fixation for Immunocytochemistry:

  • For nuclear proteins like UTP18, paraformaldehyde (4%) fixation for 15 minutes at room temperature works well

  • For better accessibility to nucleolar antigens, combine with permeabilization using 0.1% Triton X-100 for 5 minutes

  • Test methanol fixation (-20°C for 10 minutes) as an alternative if PFA yields poor results

Optimization Table for UTP18 Detection Methods:

Considerations for HRP-conjugated Antibody Detection:

• Signal Amplification Options:

  • For low abundance detection, consider using tyramide signal amplification (TSA)

  • This can increase sensitivity by 10-100 fold compared to conventional detection

  • Protocol: After primary antibody incubation, incubate with biotinylated tyramide (1:50) in amplification buffer containing 0.0015% H₂O₂ for 10 minutes

• Background Reduction Strategies:

  • Pre-incubate sections/cells with 0.3% H₂O₂ in methanol for 30 minutes to block endogenous peroxidases

  • Include 0.3M NaCl in antibody diluent to reduce non-specific ionic interactions

  • Use avidin/biotin blocking kit if using biotinylated detection systems

• Multiplexing with Other Markers:

  • For co-localization studies with nucleolar markers, use HRP-conjugated UTP18 antibody with alkaline phosphatase-conjugated second marker

  • Develop sequentially with different substrates (DAB for HRP, Fast Red for AP)

  • Alternative approach: Perform sequential tyramide labeling with different fluorophores

The optimal detection of UTP18 in nucleolar compartments typically requires careful optimization of these parameters based on the specific experimental system and questions being addressed.

How can UTP18 antibodies be used to investigate the role of UTP18 in pre-rRNA processing?

Investigating the role of UTP18 in pre-rRNA processing requires a combination of immunoprecipitation, RNA-protein interaction studies, and functional analyses. HRP-conjugated and unconjugated UTP18 antibodies can be valuable tools in these investigations.

Methodological Approaches for Studying UTP18 in pre-rRNA Processing:

• RNA Immunoprecipitation (RIP) Protocol:

  • Cross-link cells with 1% formaldehyde for 10 minutes at room temperature

  • Lyse cells in NETN buffer (20 mM Tris-HCl pH 8, 100 mM KCl, 5 mM Mg(OAc)₂, 0.5% Triton X-100, 0.1% Tween-20, plus protease inhibitors)

  • Sonicate lysate to shear chromatin (10-15 cycles of 30s on/30s off)

  • Clarify by centrifugation at 16,000× g for 10 minutes

  • Pre-clear with protein A/G beads for 1 hour at 4°C

  • Immunoprecipitate with 3-5 μg UTP18 antibody per mg of lysate overnight at 4°C

  • Isolate RNA from immunoprecipitates using TRIzol reagent

  • Analyze pre-rRNA species by northern blot or RT-qPCR

• Analysis of pre-rRNA Processing Steps:

  • Extract total RNA from cells after UTP18 knockdown/overexpression

  • Perform northern blot analysis using probes specific for various pre-rRNA intermediates

  • Key probes should target the 5'-ETS, ITS1, and ITS2 regions of the pre-rRNA

  • Quantify the ratios of precursor to mature rRNA species using phosphorimager analysis

Data Table: Expected pre-rRNA Species Accumulation After UTP18 Depletion:

• Pulse-Chase Analysis of rRNA Processing:

  • Pulse cells with ³²P-orthophosphate or methyl-methionine for 15-30 minutes

  • Chase with non-radioactive media for various time points (0, 15, 30, 60, 120 min)

  • Extract total RNA and resolve by denaturing gel electrophoresis

  • Visualize labeled RNA species by autoradiography

  • Compare processing kinetics between control and UTP18-depleted cells

• Chromatin Immunoprecipitation (ChIP) at rDNA Loci:

  • Cross-link cells with 1% formaldehyde for 10 minutes

  • Isolate nuclei and sonicate to generate chromatin fragments of ~200-500bp

  • Immunoprecipitate with UTP18 antibody

  • Analyze by qPCR using primers targeting various regions of the rDNA locus

  • Focus on the promoter, 5'-ETS, 18S, ITS1, 5.8S, ITS2, and 28S regions

Advanced Co-immunoprecipitation Strategy for UTP18 Protein Interactions:

• Perform sequential immunoprecipitation using UTP18 antibody followed by antibodies against other SSU processome components

• Analyze isolated complexes for protein composition by mass spectrometry

• Examine associated RNA species by RT-PCR or RNA sequencing

• Map interaction domains through expression of truncated UTP18 constructs

By combining these approaches, researchers can dissect the specific role of UTP18 in pre-rRNA processing, identify its direct binding sites on pre-rRNA, characterize its protein interaction network within the SSU processome , and determine the consequences of UTP18 dysfunction on ribosome biogenesis.

What troubleshooting strategies can be employed when experiencing non-specific binding with UTP18 antibody, HRP conjugated?

Non-specific binding is a common challenge when working with antibodies, including HRP-conjugated UTP18 antibodies. The following comprehensive troubleshooting guide addresses various sources of non-specificity and provides methodological solutions specific to UTP18 detection.

Systematic Troubleshooting Approach for Non-specific Binding:

• Antibody Validation and Specificity Assessment:

  • Perform peptide competition assay using the immunizing peptide (amino acids 13-152 for HRP-conjugated anti-UTP18)

  • Test antibody on UTP18 knockout/knockdown samples as negative controls

  • Compare staining patterns with different UTP18 antibodies targeting distinct epitopes

  • Verify expected molecular weight (62 kDa) in Western blots

• Optimizing Blocking Conditions:

  • Test different blocking agents:

    • 3-5% BSA in TBST (may be superior for phosphoprotein detection)

    • 5% non-fat dry milk in TBST (effective for many applications)

    • Commercial blocking buffers with proprietary formulations

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

  • Add 0.1-0.3% Triton X-100 to blocking buffer for better penetration

Optimization Table for Reducing Background in Different Applications:

• Buffer and Reagent Optimization:

  • Increase detergent concentration in wash buffers (0.1-0.3% Tween-20)

  • Add 0.1-0.5M NaCl to antibody dilution buffer to reduce ionic interactions

  • For nucleolar proteins like UTP18, add 0.1% SDS to extraction buffer to disrupt strong nucleic acid interactions

  • Use freshly prepared buffers to avoid contamination issues

• HRP-Specific Considerations:

  • Quench endogenous peroxidases thoroughly (3% H₂O₂ for 15-30 minutes)

  • Test alternative substrates (AEC instead of DAB for less sensitivity to endogenous peroxidases)

  • Reduce substrate incubation time to minimize background development

  • For Western blots, try different ECL reagent ratios or lower sensitivity detection systems

• Cross-Reactivity Mitigation for UTP18:

  • UTP18 has sequence similarity with other WD repeat-containing proteins

  • Pre-absorb antibody with recombinant WD repeat proteins

  • Use peptide-purified antibody fractions for critical applications

  • When detecting in heterologous systems, validate antibody on the target species lysate

• Sample Preparation Refinements:

  • For nucleolar proteins like UTP18, optimize nuclear extraction procedures:

    • Use hypotonic lysis followed by nuclear extraction buffer with high salt (420mM NaCl)

    • Perform stepwise extraction to separate nucleoplasmic and nucleolar fractions

  • For fixed samples, test different fixation protocols (PFA vs. methanol) to optimize epitope accessibility

  • Increase antigen retrieval stringency for formalin-fixed samples (pH 6.0 citrate buffer at 95-98°C for 30 minutes)

• Advanced Experimental Controls:

  • Implement dual-labeling with antibodies against known UTP18 interactors (Utp6, Utp21)

  • Use fluorescently-tagged UTP18 expression constructs as positive controls for localization

  • Perform parallel detection with commercial vs. in-house antibodies to validate findings

By systematically addressing these aspects of the experimental protocol, researchers can significantly reduce non-specific binding issues when working with HRP-conjugated UTP18 antibodies, thereby improving the reliability and interpretability of their data.