YPEL5 Antibody, HRP conjugated

What is YPEL5 and why is it significant for cell biology research?

YPEL5 belongs to the yippee family and serves as a component of the GID/CTLH E3 ubiquitin ligase complex. During cell cycle progression, YPEL5 demonstrates dynamic subcellular localization - found in the nucleus and centrosome during interphase, then sequentially relocating to spindle poles, mitotic spindle, and spindle midzone during mitosis, before finally transferring to the midbody during cytokinesis . This protein is particularly significant for research as it participates in cell division-related functions and contributes to preventing cell cycle exit in G1 phase, partly through the degradation of the transcription factor Hbp1 .

What are the key characteristics of HRP-conjugated antibodies in immunodetection?

Horseradish peroxidase (HRP) is a 44 kDa glycoprotein containing 6 lysine residues that can be conjugated to antibodies for various immunodetection applications . The enzyme generates visible products through chromogenic reactions with substrates such as diaminobenzidine (DAB), ABTS, or TMB in the presence of hydrogen peroxide . HRP-conjugated antibodies facilitate direct detection methods, eliminating cross-species reactivity concerns and reducing procedural complexity in time-consuming protocols by removing additional washing and separation steps .

What applications are suitable for YPEL5 antibodies with HRP conjugation?

YPEL5 antibodies with HRP conjugation are primarily designed for ELISA applications with recommended dilutions ranging from 1:500 to 1:1000 . While unconjugated YPEL5 antibodies can be used in Western Blot (1:1000-1:4000), Immunohistochemistry (1:50-1:500), and Immunofluorescence applications , the HRP-conjugated versions specifically enhance efficiency in direct detection ELISA systems through elimination of secondary antibody steps.

How should buffer conditions be optimized for working with YPEL5 HRP-conjugated antibodies?

When working with YPEL5 HRP-conjugated antibodies, buffer optimization is critical for maintaining both antibody integrity and HRP activity. The optimal conditions include:

The buffer should be free from components that interfere with HRP activity or antibody binding, including thiomersal/thimerosal, merthioloate, sodium azide, glycine, proclin, and nucleophilic components (primary amines such as amino acids or ethanolamine, and thiols such as mercaptoethanol or DTT) . For long-term storage and preservation of activity, specialized stabilizers like LifeXtend™ can protect against factors that diminish performance over time, including temperature effects and dilution-related activity loss .

What is the optimal protocol for creating custom YPEL5-HRP conjugates for specialized research applications?

For researchers requiring custom YPEL5-HRP conjugates, two primary conjugation methods are recommended:

Method 1: Periodate Oxidation Method
This approach leverages the glycoprotein nature of HRP, utilizing its polysaccharide chains in cross-linking reactions:

• Activate HRP (4 mg) by mixing with 1 ml of freshly prepared 0.1M sodium periodate for 20 minutes at room temperature to oxidize sugar moieties

• Dialyze the oxidized HRP against 1mM sodium acetate buffer (pH 4.4) overnight at 4°C

• Adjust pH to 9.0-9.5 using 0.2M sodium carbonate buffer

• Mix with purified YPEL5 antibody (typically 1-2 mg) at a 1:1 to 1:2 (HRP:antibody) molar ratio

• Incubate for 2-3 hours at room temperature

• Add sodium borohydride (4 mg/ml) to a final concentration of 0.1 mg/ml, incubate for 2 hours at 4°C

• Dialyze against PBS and validate with titration in the intended application

Method 2: Heterobifunctional Cross-Linker Method
Using sulfo-SMCC and SATA reagents for controlled conjugation:

• Activate HRP with sulfo-SMCC (10-20 fold molar excess) for 30 minutes at room temperature

• Simultaneously modify YPEL5 antibody with SATA (5-10 fold molar excess)

• Purify both activated proteins by gel filtration

• Deprotect the SATA-modified antibody with hydroxylamine

• Mix the maleimide-activated HRP with the thiolated antibody

• Incubate for 2 hours at room temperature

• Purify the conjugate and validate with titration testing

The working dilutions for custom conjugates typically range from 1:100 to 1:10,000, dependent on antibody affinity, application type, and antigen quality .

How can one verify the successful conjugation and functionality of YPEL5-HRP antibodies?

Verification of successful YPEL5-HRP conjugation should employ multiple complementary approaches:

• Spectrophotometric Analysis: Measure absorbance at 280 nm (protein) and 403 nm (HRP) to determine the protein-to-enzyme ratio in the conjugate.

• SDS-PAGE Analysis: Compare the molecular weight shift between unconjugated antibody and the HRP-conjugate (expected shift approximately 44 kDa).

• Functional Activity Testing:

  • ELISA Titration: Perform a checkerboard titration using known YPEL5-positive samples to determine optimal working dilution

  • Colorimetric Activity: Test HRP enzymatic activity using TMB substrate

  • Specificity Validation: Confirm specific binding to YPEL5 versus control proteins

• Western Blot Validation: Compare detection of YPEL5 (14 kDa) in positive control tissues (brain, testis) using both the conjugated antibody and a conventional two-step detection system with the unconjugated antibody .

How can YPEL5 HRP-conjugated antibodies be utilized to investigate YPEL5's role in the GID/CTLH E3 ubiquitin ligase complex?

YPEL5 HRP-conjugated antibodies offer distinct advantages for investigating YPEL5's role in the GID/CTLH complex through several specialized approaches:

• Co-immunoprecipitation Studies: Use YPEL5 HRP-conjugated antibodies to directly detect YPEL5 and associated proteins in co-IP experiments, enabling identification of complex components without secondary antibody steps. This is particularly valuable when examining interactions between YPEL5 and other GID/CTLH components including RanBP9 (Gid1), c17ORF39 (Gid4), Armc8 (Gid5), Twa1 (Gid8), MAEA (Gid9), and WDR26 .

• Chromatin Immunoprecipitation Analysis: Apply YPEL5 HRP-conjugated antibodies to investigate potential interactions between the GID/CTLH complex and the transcription factor Hbp1, which has been identified as a putative substrate degraded by this complex during cell cycle regulation .

• Cell Cycle Dependency Studies: Utilize direct HRP detection in immunocytochemistry to track YPEL5 subcellular localization throughout cell cycle phases, correlating localization patterns with specific GID/CTLH complex activities and substrate interactions .

• Subcellular Fractionation Analysis: Compare YPEL5 distribution and interacting partners between nuclear and cytoplasmic fractions, as research indicates the CTLH complex exhibits distinct interactomes in these compartments .

What methodological considerations are important when analyzing YPEL5 compartmentalization between nucleus and cytoplasm?

When analyzing YPEL5 compartmentalization, several critical methodological considerations should be addressed:

• Clean Subcellular Fractionation: Employ validated fractionation protocols that minimize cross-contamination between nuclear and cytoplasmic compartments. Recent research demonstrates that YPEL5 associates with the CTLH complex in both compartments but interacts with distinct factors in each location .

• Proper Controls: Include markers for both compartments (e.g., HDAC2 for nucleus, TNKS1/2 for cytoplasm) to verify fractionation quality .

• Fixation Considerations: For immunocytochemistry applications, optimize fixation conditions to preserve both nuclear and cytoplasmic epitopes, as over-fixation may mask cytoplasmic YPEL5 detection.

• Cell Cycle Synchronization: Implement cell cycle synchronization when comparing YPEL5 distributions, as its localization changes dramatically throughout the cell cycle .

• Proximity Ligation Assays: Consider using HRP-conjugated YPEL5 antibodies in proximity ligation assays (PLAs) to visualize interactions with compartment-specific binding partners in situ, following the approach used with unconjugated antibodies to confirm RanBPM association with TNKS1/2 (cytoplasm) and macroH2A1 (nucleus) .

How can researchers address data inconsistencies when YPEL5 antibody results differ between compartmental analyses?

When facing inconsistent results in YPEL5 compartmental analyses, researchers should systematically evaluate several technical and biological factors:

• Epitope Masking: YPEL5's interactions with different protein complexes may mask epitopes in compartment-specific manners. If experiencing inconsistent detection between nuclear and cytoplasmic fractions, consider:

  • Using multiple antibodies targeting different YPEL5 epitopes

  • Employing different protein extraction methods that may disrupt various protein-protein interactions

  • Validating findings with complementary approaches (e.g., tagged YPEL5 expression)

• Cell Cycle Variation: Given YPEL5's dynamic localization throughout the cell cycle, seeming inconsistencies may reflect biological variation in unsynchronized populations. Implement cell cycle synchronization protocols and conduct time-course analyses to resolve this variability .

• Cross-Reactivity Assessment: YPEL5 shares sequence homology with other YPEL family members. Confirm antibody specificity through:

  • Knockout/knockdown validation

  • Peptide competition assays

  • Testing in tissues with differential expression of YPEL family members

• Technical Validation: When data from different methodologies conflict, implement orthogonal approaches:

  • Use proximity ligation assays to validate interactions in situ

  • Combine biochemical fractionation with microscopy-based localization

  • Employ dynamic protein interaction profiling methods like TurboID

What are the technical limitations when using HRP-conjugated YPEL5 antibodies for studying protein-protein interactions?

Despite their advantages, HRP-conjugated YPEL5 antibodies present several technical limitations for protein interaction studies:

• Steric Hindrance: The large size of HRP (44 kDa) may interfere with antibody binding to YPEL5 within protein complexes, particularly the GID/CTLH complex where YPEL5 makes multiple protein contacts. This hindrance can be evaluated by:

  • Comparative binding assays between unconjugated and HRP-conjugated antibodies

  • Using alternative conjugation strategies with smaller reporter molecules

  • Testing different antibody clones with distinct epitope recognition

• Conformational Effects: HRP conjugation may alter antibody conformation, affecting binding kinetics or avidity. Researchers should:

  • Validate conjugates against unconjugated antibodies in controlled experiments

  • Test multiple conjugation protocols that attach HRP at different sites

  • Consider direct comparison with biotin-based detection systems

• Buffer Compatibility: HRP activity is sensitive to common reagents used in protein interaction studies. Avoid:

  • Reducing agents (DTT, β-mercaptoethanol) that disrupt HRP structure

  • Sodium azide which inhibits peroxidase activity

  • Detergents at concentrations that may denature the enzyme

  • High salt conditions that may alter enzyme kinetics

• Cross-linking Artifacts: Chemical conjugation methods may create artificial cross-links between antibodies or between antibody and non-target proteins. This can be minimized by:

  • Careful purification after conjugation

  • Using site-specific conjugation methods

  • Validating results with unconjugated antibodies in two-step detection protocols

How do researchers optimize proximity ligation assays (PLA) when incorporating YPEL5 HRP-conjugated antibodies?

Optimizing proximity ligation assays with YPEL5 HRP-conjugated antibodies requires several specialized considerations:

• Conjugate Modification Strategy: Standard PLA protocols may need adaptation when using HRP-conjugated antibodies. Consider:

  • Using the HRP-conjugated YPEL5 antibody with unconjugated antibodies against interaction partners

  • Implementing a modified protocol where HRP activity is blocked after primary binding but before PLA oligonucleotide ligation

  • Employing custom PLA probes specifically designed for HRP-conjugated antibodies

• Signal Discrimination: When studying proteins that change localization during cell cycle, like YPEL5, implement:

  • Cell cycle synchronization methods to enrich for specific phases

  • Co-staining with cell cycle phase markers

  • High-resolution imaging to distinguish between closely positioned structures (e.g., centrosome vs. nuclear envelope)

• Validation Controls: Include comprehensive controls to ensure signal specificity:

  • Protein depletion controls (as demonstrated in RanBPM shRNA HeLa cells)

  • Single primary antibody controls

  • Competition with unconjugated antibodies

  • Peptide blocking experiments

• Technical Optimization:

  • Adjust fixation conditions to preserve both epitope accessibility and cellular architecture

  • Optimize detergent permeabilization to balance nuclear and cytoplasmic detection

  • Calibrate antibody concentrations to achieve optimal signal-to-noise ratios

  • Consider super-resolution microscopy techniques for studying YPEL5 at subcellular structures like the midbody or centrosome

How can YPEL5 HRP-conjugated antibodies be employed in dynamic protein interaction profiling experiments?

YPEL5 HRP-conjugated antibodies can be strategically integrated into dynamic protein interaction profiling through several advanced approaches:

• Sequential Immunoprecipitation: Implement a modified ChIP-ReChIP approach where HRP-conjugated YPEL5 antibodies are used to detect secondary interactors after primary immunoprecipitation of GID/CTLH complex components. This enables identification of transient or weak interactions that may be lost in single-step protocols .

• Proximity-Dependent Biotinylation Integration: Combine HRP-conjugated antibody detection with proximity-based labeling techniques:

  • Use HRP-conjugated YPEL5 antibodies to validate interactions identified through TurboID or BioID approaches

  • Implement APEX2-based proximity labeling with HRP detection for orthogonal validation

  • Correlate interaction dynamics identified in proximity labeling with direct HRP detection

• Temporal Resolution Studies: Apply HRP-conjugated YPEL5 antibodies in time-course experiments tracking YPEL5 redistribution and protein interactions during:

  • Cell cycle progression

  • Cellular stress responses

  • Differentiation processes

  • Metabolic state changes related to GID/CTLH complex activity

• Comparative Interactome Analysis: Utilize HRP-conjugated YPEL5 antibodies to compare interaction profiles between:

  • Nuclear vs. cytoplasmic fractions

  • Different cell types or tissues

  • Normal vs. disease states

  • Wild-type vs. mutant YPEL5 models

What considerations are important when developing assays to investigate YPEL5's role in ubiquitin-dependent protein degradation?

When developing assays to investigate YPEL5's role in ubiquitin-dependent protein degradation as part of the GID/CTLH complex, researchers should consider:

• Substrate Identification Strategy: YPEL5 HRP-conjugated antibodies can facilitate identification of potential substrates through:

  • Immunoprecipitation of YPEL5 followed by proteomics analysis in proteasome-inhibited conditions

  • Western blot screening of candidate substrates in YPEL5-depleted vs. control cells

  • Direct detection in cell-free ubiquitination assays

• E3 Ligase Activity Assays: When reconstituting in vitro ubiquitination assays:

  • Include appropriate E1 and E2 enzymes (specifically UBE2H, which is selectively accepted by the GID/CTLH complex)

  • Verify the presence of all critical complex components (YPEL5, RanBP9, RMND5a/b, etc.)

  • Monitor both substrate ubiquitination and degradation

  • Compare wild-type vs. mutant YPEL5 incorporation into the complex

• Cell-Based Degradation Assays: Design experiments that can detect changes in substrate stability:

  • Focus on known substrates like Hbp1 or identify new substrates through proteomic approaches

  • Implement cycloheximide chase experiments with and without proteasome inhibitors

  • Use fluorescent protein degron systems coupled with YPEL5 manipulation

  • Correlate degradation kinetics with cell cycle progression

• Complex Assembly Analysis: Determine how YPEL5 contributes to GID/CTLH complex formation and function:

  • Assess whether YPEL5 is required for complex assembly or stability

  • Investigate if YPEL5 contributes to substrate recognition or specificity

  • Determine if YPEL5 modulates E2 enzyme recruitment or activity

  • Examine potential redundancy with other complex components or paralogues

What are the recommended positive control tissues for validating YPEL5 HRP-conjugated antibodies?

Based on validated expression patterns, the following tissues and samples serve as optimal positive controls for YPEL5 antibody validation:

For immunohistochemistry applications, antigen retrieval with TE buffer (pH 9.0) is recommended, though citrate buffer (pH 6.0) may serve as an alternative . When working with HRP-conjugated antibodies in these tissues, researchers should include appropriate controls for endogenous peroxidase activity.

What are the critical quality control parameters for evaluating YPEL5 HRP-conjugated antibodies?

Comprehensive quality control for YPEL5 HRP-conjugated antibodies should include evaluation of the following parameters:

• Antibody Specificity:

  • Single band detection at 14 kDa in Western blot

  • Anticipated subcellular localization pattern in immunocytochemistry

  • Signal reduction or elimination in YPEL5 knockdown/knockout models

  • Absence of cross-reactivity with other YPEL family members

• HRP Conjugation Efficiency:

  • Molar ratio of HRP to antibody (optimal range: 2-4 HRP molecules per antibody)

  • Retention of antibody binding capacity post-conjugation

  • Preserved HRP enzymatic activity

  • Minimal aggregation or precipitation

• Functional Performance:

  • Sensitivity (limit of detection for YPEL5 protein)

  • Dynamic range of detection

  • Signal-to-noise ratio in application-specific conditions

  • Lot-to-lot consistency in performance metrics

  • Stability under storage and working conditions

• Application-Specific Validation:

  • Titration curves to determine optimal working dilution (typically 1:500-1:1000 for ELISA)

  • Background assessment in negative control samples

  • Comparison against unconjugated antibody plus secondary detection

  • Performance in the presence of potential interfering substances

These parameters should be systematically documented to ensure reproducible results across experimental applications and research contexts .