LEO1 Antibody, FITC conjugated

What is LEO1 and what are its biological functions?

LEO1 (RNA polymerase-associated protein LEO1) is a 666 amino acid protein belonging to the LEO1 family. It functions as a critical component of the PAF1 complex (PAF1C), which plays multiple roles during transcription by RNA polymerase II . LEO1 is implicated in:

• Regulation of development and maintenance of embryonic stem cell pluripotency

• Histone modifications including ubiquitination of histone H2B and methylation of histone H3 'Lys-4' (H3K4me3)

• Recruitment of the RNF20/40 E3 ubiquitin-protein ligase complex

• mRNA 3' end formation through association with cleavage and poly(A) factors

• Connection of PAF1C to Wnt signaling

The protein has a calculated molecular weight of 75 kDa, but due to post-translational modifications, it is typically observed at approximately 105 kDa in Western blot applications .

What is FITC conjugation and why is it used for antibody labeling in LEO1 research?

FITC (Fluorescein isothiocyanate) conjugation is a chemical process that attaches fluorescein molecules to antibodies via primary amines (typically lysine residues). This conjugation enables direct visualization of target proteins without requiring secondary antibodies .

For LEO1 research, FITC-conjugated antibodies offer several advantages:

• Direct detection of LEO1 in applications such as immunofluorescence microscopy and flow cytometry

• Elimination of potential cross-reactivity issues associated with secondary antibodies

• Capability for multicolor imaging when combined with other fluorophores

• Efficient visualization of nuclear proteins like LEO1 that function in transcriptional complexes

Typically, between 3-6 FITC molecules are conjugated to each antibody; higher conjugations can result in solubility problems and internal quenching that reduces brightness .

How do the spectral properties of FITC impact experimental design for LEO1 localization studies?

The spectral properties of FITC determine important parameters for experimental design when studying LEO1 localization:

When designing multiplexed experiments with LEO1-FITC antibodies, researchers must consider spectral overlap with other fluorophores. FITC emission may bleed into PE channels, requiring proper compensation in flow cytometry or selection of spectrally distinct fluorophores (such as Alexa Fluor 594-conjugated LEO1 antibodies) for co-localization studies .

What experimental applications have been validated for FITC-conjugated LEO1 antibodies?

Based on the search results, FITC-conjugated LEO1 antibodies have been validated for several applications:

For optimal results in immunohistochemistry applications, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may serve as an alternative .

What cell and tissue types have been validated for LEO1 detection?

LEO1 expression has been confirmed in numerous cell and tissue types, which provides valuable information for experimental design:

For Western blot applications:

• Human cell lines: HeLa, HL-60, Jurkat, HepG2, HT-29, A549, HEK-293, LNCaP, MCF-7

• Mouse tissues: heart, brain, skeletal muscle

• Human tissues: placenta

For immunohistochemistry:

• Human colon tissue has been positively validated

When designing experiments to study LEO1 in novel tissue types, researchers should begin with these validated samples as positive controls to establish staining protocols before investigating experimental tissues .

How should researchers optimize antigen retrieval for LEO1 immunostaining?

Antigen retrieval optimization is critical for successful LEO1 immunostaining due to potential epitope masking during fixation:

• Primary recommendation: Use TE buffer pH 9.0 for heat-induced epitope retrieval (HIER)

• Alternative approach: Citrate buffer pH 6.0 may be effective for some tissue types

• For paraffin-embedded tissue sections (FFPE): Tris-EDTA pH 9.0 is specifically recommended

Researchers should conduct a systematic comparison of retrieval methods when working with new tissue types or fixation protocols. This comparison should include:

• pH gradient testing (pH 6.0, 8.0, and 9.0)

• Retrieval time optimization (10-30 minutes)

• Temperature assessment (95-100°C)

The efficacy of antigen retrieval can be tissue-dependent, and optimization may significantly improve signal-to-noise ratio in LEO1 detection .

How does the FITC labeling index affect LEO1 antibody performance?

The FITC labeling index (number of FITC molecules per antibody) critically impacts antibody performance in several ways:

Research findings indicate that FITC-labeled antibodies used in tissue cross-reactivity studies should be carefully selected from several differently labeled preparations to minimize decreased binding affinity while achieving appropriate sensitivity . When investigating LEO1 in complex tissues, researchers should consider testing multiple FITC-labeled LEO1 antibody preparations with different labeling indices to determine optimal performance for their specific application .

What storage conditions maximize stability of FITC-conjugated LEO1 antibodies?

Proper storage is essential for maintaining the functionality of FITC-conjugated LEO1 antibodies:

Critical notes:

• Avoid repeated freeze-thaw cycles as this may denature the antibody

• Storage in frost-free freezers is not recommended due to temperature fluctuations

• For antibodies in smaller volumes (20μl), note that preparations may contain 0.1% BSA as a stabilizer

How can researchers verify successful FITC conjugation to LEO1 antibodies?

Verification of successful FITC conjugation is essential before proceeding with experiments. Researchers can employ several methods:

• Spectrophotometric analysis:

  • Measure absorbance at 280 nm (protein) and 495 nm (FITC)

  • Calculate F/P (fluorophore to protein) ratio using the formula:
    F/P ratio = [A495 × dilution factor × MW of protein] / [195,000 × protein concentration (mg/ml)]

  • Optimal F/P ratio typically ranges from 3-6 for LEO1 antibodies

• Preliminary application testing:

  • Perform a small-scale experiment using positive control samples (e.g., HeLa cells for IF/ICC)

  • Include both positive controls (known LEO1-expressing cells) and negative controls (cells with LEO1 knockdown or tissues known not to express LEO1)

• Fluorescence-based quality control:

  • Use conjugation check kits that allow confirmation of conjugation in one step

  • Note that these kits are only suitable for qualitative verification of IgG antibodies

The most practical approach is combining spectrophotometric analysis with application-specific validation on known positive controls to ensure both technical conjugation success and functional antibody activity .

How does FITC conjugation affect epitope binding and quantitative analysis of LEO1?

FITC conjugation can significantly impact epitope binding and subsequent quantitative analysis of LEO1 through several mechanisms:

• Steric hindrance effects:

  • FITC molecules bound to lysine residues near the antigen-binding site may physically interfere with antigen recognition

  • This may reduce binding affinity by 10-40% depending on the labeling index

  • For LEO1 detection, this can be particularly problematic when studying protein-protein interactions within the PAF1 complex

• Quantitative implications:

  • Signal intensity may not linearly correlate with protein abundance due to variable conjugation efficiency

  • Comparative quantitative studies should use the same antibody lot to minimize variation

  • Absolute quantification requires calibration with known standards of similar F/P ratio

• Binding kinetics alterations:

  • FITC conjugation typically reduces the on-rate (association constant) while minimally affecting the off-rate

  • This results in higher Kd values (lower affinity) that may require adjusted incubation times

  • For LEO1, which has a calculated MW of 75 kDa but runs at 105 kDa due to modifications, binding kinetics can be particularly affected

Researchers performing quantitative analysis of LEO1 expression should conduct careful validation studies comparing FITC-conjugated antibodies with unconjugated antibodies followed by indirect detection to establish correction factors for quantitative measurements .

What controls should be included when studying LEO1 using FITC-conjugated antibodies?

Comprehensive experimental design for LEO1 studies requires multiple control types:

Additionally, when studying LEO1's role in the PAF1 complex, coordinate expression analysis of other complex members (PAF1, CTR9, CDC73, RTF1) provides valuable context for functional studies .

What methodological approaches enable studying LEO1's functional role in transcriptional regulation?

Advanced research into LEO1's functional role in transcriptional regulation requires sophisticated methodological approaches:

• Chromatin immunoprecipitation (ChIP) using FITC-conjugated LEO1 antibodies:

  • Enables identification of LEO1-bound genomic regions

  • Can be coupled with sequencing (ChIP-seq) for genome-wide binding profiles

  • Requires optimization of crosslinking conditions due to LEO1's role in large protein complexes

• Co-immunoprecipitation studies:

  • Investigate LEO1's interactions within the PAF1 complex

  • The IP application has been validated for LEO1 antibodies

  • Note: FITC conjugation may affect protein-protein interaction detection efficiency

• Histone modification analysis in LEO1-depleted systems:

  • LEO1 functions in histone H3K4 trimethylation

  • Quantitative assessment of H3K4me3 levels following LEO1 knockdown/knockout

  • Correlation with transcriptional activity at specific loci

• Functional analysis in developmental contexts:

  • RNAi-mediated reduction of LEO1 affects gene expression and development in Drosophila

  • FITC-conjugated antibodies can track LEO1 localization during developmental transitions

  • Connection to Notch signaling pathways through histone modification pathways

These methodological approaches should consider that the LEO1 protein functions within multi-protein complexes that regulate transcription through histone modifications, and experimental designs should account for these complex interactions .

How can researchers address non-specific staining when using FITC-conjugated LEO1 antibodies?

Non-specific staining is a common challenge with FITC-conjugated antibodies, particularly those with high labeling indices. Effective troubleshooting approaches include:

Research demonstrates that antibodies with higher FITC labeling indices are more likely to yield non-specific staining, and careful selection from differently labeled preparations is recommended . For LEO1 detection in tissues with high autofluorescence, researchers should consider alternative conjugates (e.g., Alexa Fluor 594) .

How should researchers interpret discrepancies between predicted and observed molecular weights of LEO1?

LEO1 presents an interesting case where the calculated molecular weight (75 kDa) differs significantly from the observed weight in Western blot applications (105 kDa) . When encountering such discrepancies:

• Verify identity through multiple approaches:

  • Use multiple antibodies targeting different epitopes of LEO1

  • Confirm specificity through knockdown/knockout controls

  • Perform mass spectrometry analysis of immunoprecipitated bands

• Consider post-translational modifications:

  • LEO1's role in transcriptional regulation suggests potential phosphorylation sites

  • SUMOylation may occur in nuclear proteins like LEO1

  • Glycosylation can significantly alter migration patterns

• Evaluate technical factors:

  • Gel percentage and running conditions can affect migration

  • Protein standards used for calibration

  • Sample preparation methods (denaturing conditions)

The published literature specifically notes that the modified LEO1 protein runs at approximately 105 kDa despite its 75 kDa calculated weight (PMID: 15632063) . This consistent observation across multiple studies suggests authentic post-translational modifications rather than technical artifacts or non-specific binding.