YPEL5 Antibody, Biotin conjugated

What is YPEL5 and what are its key biological functions?

YPEL5 (yippee-like 5) is a 121-amino acid protein with a molecular mass of approximately 13.8 kDa that belongs to the Yippee protein family. It functions as a component of the CTLH E3 ubiquitin-protein ligase complex, which selectively accepts ubiquitin from UBE2H and mediates ubiquitination and subsequent proteasomal degradation of the transcription factor HBP1. The protein is found in both nuclear and cytoplasmic compartments and is widely expressed across multiple tissue types . All five members of the yippee-like protein family, including YPEL5, are known to localize to the centrosome or mitotic spindle, suggesting involvement in cell division processes . YPEL5 has been identified as forming recurrent reciprocal RNA chimeras with PPP1CB in chronic lymphocytic leukemia (CLL), which may implicate it in certain cancer pathways .

What experimental applications are biotin-conjugated YPEL5 antibodies validated for?

Biotin-conjugated YPEL5 antibodies are validated for several common experimental techniques in molecular and cellular biology research. The primary validated application is Western Blot analysis (WB) , while some antibody products may also be confirmed for use in enzyme-linked immunosorbent assay (ELISA), immunocytochemistry (ICC), and immunofluorescence (IF) procedures . These applications allow researchers to detect and quantify YPEL5 protein expression in various experimental contexts, from protein extracts to intact cells. When selecting a biotin-conjugated YPEL5 antibody for research, it is critical to verify that the specific product has been validated for your intended application.

How do the molecular weights observed in experimental detection of YPEL5 compare to theoretical predictions?

An interesting observation in YPEL5 research is the discrepancy between the calculated and experimentally observed molecular weights. While the calculated molecular weight for YPEL5 is 13,842 Da (approximately 13.8 kDa) based on its amino acid sequence , experimental detection using antibodies has revealed bands at approximately 68 kDa in Western blot applications . This significant difference could be attributed to post-translational modifications, protein complexes that remain stable during sample preparation, or cross-reactivity with related proteins. Researchers should be aware of this discrepancy when interpreting their results and consider additional validation techniques such as mass spectrometry or knockout controls to confirm specificity for the target protein.

How should biotin-conjugated antibodies be stored and handled to maintain optimal activity?

Biotin-conjugated YPEL5 antibodies require specific storage and handling protocols to preserve their activity. Most manufacturers recommend storing these antibodies at -20°C for long-term preservation (up to one year) or at 4°C for short-term storage (up to three months) . It is crucial to avoid repeated freeze-thaw cycles as these can significantly diminish antibody activity through protein denaturation. When handling the antibody, researchers should minimize exposure to prolonged high temperatures and work quickly on ice when possible. Most biotin-conjugated YPEL5 antibodies are supplied in phosphate-buffered saline (PBS) containing 0.02% sodium azide as a preservative . Always refer to the manufacturer's specific recommendations for the product you are using, as formulations may vary.

What are the optimal dilution ratios for biotin-conjugated YPEL5 antibodies in different applications?

The optimal dilution ratios for biotin-conjugated YPEL5 antibodies vary depending on the specific application and the antibody concentration. For Western blot applications, typical dilutions range from 1:500 to 1:2000, depending on the specific antibody concentration and sensitivity requirements. For immunocytochemistry and immunofluorescence, dilutions of 1:100 to 1:500 are commonly used, while ELISA applications may require dilutions from 1:1000 to 1:10,000 .

When working with a new biotin-conjugated YPEL5 antibody, it is advisable to perform a titration experiment with multiple dilutions to determine the optimal concentration that provides the best signal-to-noise ratio for your specific experimental conditions and detection system. This approach will help minimize background while maintaining sensitivity for the target protein.

How can researchers validate the specificity of biotin-conjugated YPEL5 antibodies in their experimental systems?

Validating antibody specificity is crucial for reliable research results. For biotin-conjugated YPEL5 antibodies, several approaches can be employed:

• Positive and negative controls: Include samples known to express YPEL5 (positive control) and those that do not (negative control). YPEL5 is widely expressed across many tissues, making it important to use appropriate controls .

• Knockdown/knockout validation: Use YPEL5 siRNA knockdown or CRISPR-Cas9 knockout samples to confirm that the detected signal decreases or disappears accordingly.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide used to generate it. If the antibody is specific, this should block the detection signal .

• Cross-species reactivity check: Verify the antibody performance across species. The biotin-conjugated YPEL5 antibody (LS-C462478) has demonstrated reactivity with human, mouse, rat, and numerous other species, which can be useful for comparative studies .

• Molecular weight verification: Be aware that while the calculated molecular weight of YPEL5 is 13.8 kDa, it may be detected at approximately 68 kDa in some experimental conditions . This discrepancy should be considered when evaluating antibody specificity.

How does biotin conjugation affect YPEL5 antibody performance in multiplexed immunoassays?

First, biotin interference can significantly impact assay validity. High endogenous biotin levels in samples can compete with biotin-conjugated antibodies for streptavidin binding sites, potentially leading to false negative results . To mitigate this, researchers should:

• Pre-test samples for biotin levels

• Include biotin-free controls

• Consider biotin depletion steps for problematic samples

• Implement standard curves with known concentrations of biotin to quantify interference effects (as shown in the table below)

Second, for multiplex detection involving YPEL5 along with other targets, researchers should verify antibody compatibility and optimize detection parameters to prevent cross-reactivity and signal spillover between detection channels .

What strategies can address inconsistent YPEL5 detection in CLL research using biotin-conjugated antibodies?

Chronic lymphocytic leukemia (CLL) research involving YPEL5 presents unique challenges due to the formation of chimeric transcripts between YPEL5 and PPP1CB genes . When using biotin-conjugated YPEL5 antibodies in this context, researchers should implement several specialized strategies:

• Targeted epitope selection: Since YPEL5 forms chimeric transcripts with PPP1CB in CLL, select antibodies that target epitopes either preserved or lost in these fusion events. Antibodies targeting the N-terminus of YPEL5 (like LS-C462478) may be particularly valuable as they can distinguish between wild-type and chimeric forms.

• Dual detection approach: Implement simultaneous detection of both YPEL5 and PPP1CB to identify potential chimeric proteins. This can be accomplished using differentially labeled secondary antibodies against a biotin-conjugated YPEL5 antibody and another antibody targeting PPP1CB.

• Cell population isolation: As demonstrated in CLL research, YPEL5/PPP1CB chimeras were expressed specifically in CD19-enriched B-cell fractions but not in CD13/33-enriched granulocyte fractions . Therefore, proper cell isolation procedures are critical before antibody staining to ensure accurate interpretation of results.

• Complementary validation with nucleic acid analysis: Confirm protein-level findings with RT-PCR or RNA-seq data to verify the presence of YPEL5/PPP1CB chimeric transcripts, especially when unexpected results are obtained with antibody detection .

How can researchers distinguish between normal and aberrant YPEL5 expression patterns using biotin-conjugated antibodies?

Distinguishing normal from aberrant YPEL5 expression is critical for cancer and developmental biology research. Biotin-conjugated YPEL5 antibodies can be effectively employed for this purpose through several methodological approaches:

• Quantitative expression analysis: Implement quantitative Western blotting or flow cytometry with biotin-conjugated YPEL5 antibodies and streptavidin-conjugated fluorophores to precisely measure expression levels. In CLL, for example, YPEL5 shows significantly higher expression compared to other tissues, suggesting lineage or tissue-specific promoter activation .

• Subcellular localization mapping: YPEL5 normally localizes to the centrosome or mitotic spindle as well as in nuclear and cytoplasmic compartments . Using high-resolution confocal microscopy with biotin-conjugated YPEL5 antibodies and streptavidin-conjugated fluorophores, researchers can determine if YPEL5 shows abnormal localization patterns in disease states.

• Co-localization with cell cycle markers: Since YPEL5 is associated with centrosomes and mitotic spindles, co-staining with cell cycle markers can reveal whether aberrant YPEL5 expression correlates with cell cycle dysregulation. This can be particularly relevant for cancer research applications.

• Comparative tissue analysis: Create expression profiles across multiple tissue types or cell lines using standardized detection protocols with biotin-conjugated YPEL5 antibodies. This establishes baseline expression patterns for comparison with potential disease-associated variations.

What is the optimal protocol for using biotin-conjugated YPEL5 antibodies in fluorescent immunohistochemistry?

For optimal fluorescent immunohistochemistry with biotin-conjugated YPEL5 antibodies, the following protocol is recommended:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde for 24 hours

  • Process and embed in paraffin or prepare frozen sections (5-7 μm)

  • For paraffin sections, perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 20 minutes

• Blocking and antibody incubation:

  • Block endogenous biotin using a commercial biotin blocking kit to prevent background

  • Block nonspecific binding with 5% normal serum from the same species as the secondary detection reagent

  • Incubate with biotin-conjugated YPEL5 antibody (recommended dilution 1:100-1:200) overnight at 4°C

  • Wash thoroughly (3 x 5 minutes) with PBS containing 0.1% Tween-20

• Detection:

  • Incubate with fluorophore-conjugated streptavidin (e.g., streptavidin-Alexa Fluor 488 or 594) at 1:500 dilution for 1 hour at room temperature

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

  • Mount in anti-fade mounting medium

• Controls:

  • Include a biotin-blocking control to confirm elimination of endogenous biotin signal

  • Include a secondary-only control (omitting primary antibody) to assess background

  • Consider dual staining with centrosome markers (e.g., γ-tubulin) to confirm specific YPEL5 localization

How can researchers minimize biotin interference when using biotin-conjugated YPEL5 antibodies?

Biotin interference is a significant concern when using biotin-conjugated antibodies, including those targeting YPEL5. This interference occurs when endogenous biotin in samples competes with the biotin-conjugated antibody for binding to detection reagents like streptavidin . To minimize this issue:

• Sample pre-treatment methods:

  • Implement a biotin depletion step using streptavidin-coated beads or columns

  • For serum samples, consider precipitation methods to remove free biotin

  • Use commercially available biotin blocking kits before applying biotin-conjugated antibodies

• Assay design modifications:

  • Include calibration controls with known biotin concentrations (ranging from 20-5250 ng/mL) to quantify potential interference

  • Consider a serial dilution approach to identify and correct for interference effects

  • For critical samples, run parallel assays using both biotin-conjugated and non-biotin-conjugated antibodies to validate results

• Alternative detection strategies:

  • In cases of severe biotin interference, consider using directly fluorophore-conjugated secondary antibodies instead of the biotin-streptavidin system

  • Implement wash steps with higher stringency (increased salt concentration or detergent) to reduce nonspecific binding

• Quantitative correction:

  • Based on calibration data, develop a mathematical correction factor to adjust for biotin interference in your specific experimental system

What are the recommended approaches for dual staining with biotin-conjugated YPEL5 antibodies in co-localization studies?

Dual staining for co-localization studies involving biotin-conjugated YPEL5 antibodies requires careful planning to avoid cross-reactivity and ensure specific detection. Based on YPEL5's known functions and localizations, the following approaches are recommended:

• Sequential staining protocol:

  • Perform YPEL5 staining first with biotin-conjugated antibody

  • Block all remaining biotin binding sites with excessive free biotin

  • Proceed with the second primary antibody (non-biotinylated) and appropriate secondary detection

• Recommended co-staining partners for functional studies:

  • UBE2H and CTLH complex components to study YPEL5's role in the E3 ubiquitin-protein ligase complex

  • HBP1 transcription factor to investigate regulatory relationships

  • Centrosome markers (γ-tubulin) to confirm subcellular localization

  • PPP1CB to investigate potential chimeric relationships in CLL samples

• Fluorophore selection to minimize spectral overlap:

  • For biotin-conjugated YPEL5 detection: Use streptavidin-Alexa Fluor 488 (green)

  • For co-stained proteins: Use fluorophores with minimal spectral overlap, such as Alexa Fluor 594 (red) or Alexa Fluor 647 (far-red)

  • Include single-stained controls to establish proper compensation when using confocal microscopy

• Advanced microscopy techniques:

  • Implement super-resolution microscopy (STED, STORM) for precise co-localization analysis

  • Use photobleaching correction algorithms for quantitative co-localization studies

  • Consider live-cell imaging with complementary fluorescent protein fusions to validate antibody-based co-localization findings

How can biotin-conjugated YPEL5 antibodies be utilized in cancer research beyond CLL studies?

Biotin-conjugated YPEL5 antibodies have significant potential in broader cancer research due to YPEL5's emerging roles in cellular processes and disease contexts:

• Tumor tissue microarray screening: The wide species reactivity and specificity of biotin-conjugated YPEL5 antibodies make them valuable for high-throughput screening of YPEL5 expression across multiple tumor types. This approach can identify cancer types where YPEL5 is dysregulated, extending beyond the established association with CLL .

• Ubiquitination pathway investigation: Given YPEL5's role in the CTLH E3 ubiquitin-protein ligase complex , biotin-conjugated antibodies can be used to study how aberrations in this pathway contribute to cancer progression. Researchers can investigate:

  • Changes in YPEL5 expression affecting the degradation of HBP1 transcription factor

  • Altered interactions between YPEL5 and other components of the ubiquitination machinery

  • The impact of therapeutic agents targeting the ubiquitin-proteasome system on YPEL5 function

• Cell cycle and centrosome abnormality assessment: Since YPEL5 localizes to centrosomes and mitotic spindles , biotin-conjugated antibodies can be used to investigate the relationship between YPEL5 and centrosome amplification, a common feature of many cancers.

• Biomarker potential evaluation: Systematic studies correlating YPEL5 expression patterns (detected using biotin-conjugated antibodies) with clinical outcomes across multiple cancer types could identify its potential as a prognostic or predictive biomarker.

What approaches should be used to investigate YPEL5's role in the CTLH E3 ubiquitin-protein ligase complex?

Investigating YPEL5's functional role in the CTLH E3 ubiquitin-protein ligase complex requires integrated methodological approaches:

• Co-immunoprecipitation studies:

  • Use biotin-conjugated YPEL5 antibodies for pulldown experiments followed by streptavidin-coated beads

  • Analyze co-precipitated proteins by mass spectrometry to identify novel interactions within the CTLH complex

  • Verify interactions by reverse co-IP with antibodies to other complex components

  • Compare complex composition in normal versus disease states to identify pathological alterations

• Functional ubiquitination assays:

  • Implement in vitro ubiquitination assays using purified components including YPEL5, UBE2H, and potential substrates

  • Use cell-based ubiquitination assays with overexpression or knockdown of YPEL5 to assess its requirement for substrate degradation

  • Monitor HBP1 transcription factor levels and ubiquitination status as a readout of YPEL5 function

• Structure-function analysis:

  • Generate domain-specific YPEL5 mutants and use biotin-conjugated antibodies to assess their incorporation into the CTLH complex

  • Map the regions of YPEL5 required for complex formation versus substrate recognition

  • Implement FRET or PLA (proximity ligation assay) with biotin-conjugated YPEL5 antibodies to examine protein-protein interactions in situ

• Substrate identification approach:

  • Combine YPEL5 manipulation (overexpression, knockdown) with global proteomics to identify proteins whose stability is YPEL5-dependent

  • Validate candidates using cycloheximide chase experiments and ubiquitination assays

  • Use biotin-conjugated YPEL5 antibodies to confirm direct interactions with newly identified substrates

How can researchers adapt protocols for biotin-conjugated YPEL5 antibodies when working with difficult sample types?

Working with challenging sample types requires specific adaptations to standard protocols for biotin-conjugated YPEL5 antibodies:

• Formalin-fixed paraffin-embedded (FFPE) tissues:

  • Implement extended antigen retrieval (25-30 minutes) using high-temperature citrate buffer (pH 6.0)

  • Consider tyramide signal amplification (TSA) to enhance detection sensitivity

  • Use a biotin blocking kit followed by longer primary antibody incubation (overnight at 4°C)

  • Increase the concentration of the biotin-conjugated YPEL5 antibody (1:50-1:100 dilution)

  • Evaluate multiple detection systems to optimize signal-to-noise ratio

• Low abundance samples (rare cell populations):

  • Implement cell enrichment techniques prior to antibody application

  • For flow cytometry, consider using fluorescent cell sorting to concentrate target cells before staining

  • Extend incubation times for the biotin-conjugated antibody (up to 24 hours at 4°C)

  • Use signal amplification through multi-layer detection systems

• Highly autofluorescent tissues (brain, liver):

  • Pretreat sections with Sudan Black B (0.1-0.3%) to reduce lipofuscin autofluorescence

  • Use confocal microscopy with spectral unmixing to separate autofluorescence from specific signal

  • Consider non-fluorescent detection methods like chromogenic detection with streptavidin-HRP

  • Implement computational approaches for autofluorescence subtraction during image analysis

• Frozen tissue microarrays:

  • Fix sections briefly (10 minutes) in cold acetone rather than paraformaldehyde

  • Block with protein-free blocking buffer to reduce background

  • Optimize antibody concentration through titration experiments (typically using higher dilutions than for FFPE)

  • Consider direct detection methods to reduce background associated with streptavidin-biotin amplification