EED Antibody

Basic Research Questions

• What is the optimal storage condition for EED antibodies to maintain long-term functionality?

EED antibodies should be stored according to manufacturer specifications, with most requiring storage at -20°C to -70°C for long-term stability. Data indicates optimal storage conditions include:

Research shows that repeated freeze-thaw cycles significantly decrease antibody effectiveness . For optimal preservation, aliquot antibodies upon receipt, store in a manual defrost freezer, and avoid prolonged exposure to high temperatures .

• What applications are validated for commercially available EED antibodies?

EED antibodies are validated for multiple experimental applications with varying success rates:

When selecting an EED antibody, verify that it has been specifically validated for your application and experimental system . Manufacturer validation data typically includes positive controls such as Jurkat, HT-29, HepG2, and HeLa cell lines .

• What is the expected molecular weight of EED protein in Western blot analyses?

The expected molecular weight for EED varies based on isoform detection and experimental conditions:

Research indicates that EED displays multiple bands between 50-70 kDa in reducing conditions, with a specific band often detected at approximately 58 kDa when using certain antibodies . The variability is due to alternative start sites, including one that generates a 69 kDa isoform, and post-translational modifications .

• How should I determine the optimal antibody dilution for my specific experiment?

Determining optimal dilution requires systematic titration:

• Begin with the manufacturer's recommended dilution range (e.g., 1:500-1:2000 for WB)

• Perform a dilution series experiment using positive control samples

• Evaluate signal-to-noise ratio at each dilution

• Consider cell/tissue-specific expression levels of EED

Best practices indicate that optimal dilutions should be determined independently for each application and experimental system . For specialized applications like ChIP-Seq, starting with ~4 μl per reaction is recommended, followed by optimization .

• What are suitable positive controls for EED antibody validation?

Several validated cell lines and tissues serve as reliable positive controls:

For rigorous validation, include a negative control such as EED knockout samples, which should show no band when probed with anti-EED antibody . Specialized applications may require tissue-specific controls.

Advanced Research Questions

• How do different epitope-targeting EED antibodies affect detection of protein-protein interactions?

Epitope selection critically influences detection of EED's interactions with PRC1 and PRC2 complexes:

Research demonstrates that antibodies targeting different EED epitopes reveal distinct protein interactions. Anti-EED (aa186-304) pulled down both PRC2 components (EZH2, SUZ12) and PRC1 components (BMI1, RING1B), while anti-EED (aa429-441) pulled down only PRC1 components . This explains why previous studies using N-terminal tagged EED constructs failed to detect EED:PRC1 interactions . For comprehensive interaction studies, use antibodies targeting central regions or employ multiple antibodies targeting different epitopes.

• What strategies can minimize batch-to-batch variability issues with EED antibodies?

Batch-to-batch variability poses significant challenges, particularly with polyclonal antibodies:

Research indicates that batch variability is particularly problematic with polyclonal antibodies . When critical for longitudinal studies, validate each new batch against previous batches using identical samples and protocols. Document batch numbers in publications to facilitate reproducibility across research groups .

• How do fixation methods affect EED antibody performance in immunohistochemistry?

Fixation significantly impacts epitope accessibility and antibody performance:

For optimal IHC results with FFPE tissues, heat-induced epitope retrieval using TE buffer at pH 9.0 is generally recommended . For tissues with high background, citrate buffer at pH 6.0 may provide better signal-to-noise ratio. Perform side-by-side comparisons of different retrieval methods to determine optimal conditions for specific antibody-tissue combinations.

• What are the key considerations for using EED antibodies in co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) with EED antibodies requires careful optimization:

Research shows that antibody binding at the EED N-terminus may hinder EED:PRC1 interaction detection . When studying potential new interaction partners, use antibodies targeting central regions (e.g., aa186-304) that preserve multiple interaction interfaces. Validation studies should include multiple washing stringencies to confirm specific interactions .

• What validation approaches should be employed to ensure EED antibody specificity for ChIP and ChIP-Seq experiments?

Rigorous validation is critical for chromatin immunoprecipitation applications:

For ChIP-Seq applications, validation should include demonstration of enrichment at known EED target genes such as HOXC8, HOXA9, MYT1, and CDKN2A . The most rigorous validation combines knockout controls with the use of multiple antibodies targeting different epitopes . Publications should thoroughly document validation methods to enhance reproducibility .