EGLN2 Antibody

What is EGLN2 and why is it important in research?

EGLN2 (also known as PHD1, HPH-3, HIFPH1) is a 43-48 kDa prolyl hydroxylase family member primarily localized in the nucleus . It functions as an oxygen sensor by hydroxylating proline residues on HIF-1α, leading to HIF-1α ubiquitylation and degradation under normoxic conditions . Beyond oxygen sensing, EGLN2 regulates mitochondrial function in breast cancer through a HIF1/2α-independent mechanism . Its dysregulation has been implicated in various diseases including cancer, cardiovascular disorders, and neurodegenerative conditions . Recent research has demonstrated EGLN2's role in forming activator complexes with PGC1α and NRF1 on chromatin to promote transcription of genes like ferridoxin reductase (FDXR) .

What molecular characteristics should be considered when selecting an EGLN2 antibody?

When selecting an EGLN2 antibody, researchers should consider:

• Recognition epitope: Antibodies targeting different regions of EGLN2 may yield varying results. Some antibodies are generated against synthetic peptides corresponding to sequences within amino acids 200-300 of human EGLN2 .

• Species reactivity: Many commercial antibodies react with human, mouse, and rat EGLN2 .

• Application compatibility: Verify validation for specific applications (WB, IP, ELISA, ChIP-seq, etc.) .

• Molecular weight detection: EGLN2 has a calculated MW of 44 kDa but is typically observed at 43-48 kDa in Western blots .

• Cellular localization: Consider whether the antibody can detect both nuclear and cytoplasmic EGLN2, as the protein localizes in both compartments with enrichment in nuclear and chromatin-bound fractions upon hypoxia exposure .

How should Western blot conditions be optimized for EGLN2 detection?

For optimal EGLN2 detection in Western blot applications:

• Sample preparation: Use appropriate cell lysis buffers compatible with nuclear proteins.

• Protein amount: Load 20-50 μg of total protein per lane.

• Dilution range: Use antibody dilutions between 1:500-1:2000 .

• Controls: Include positive controls such as lysates from HeLa, A-549, HepG2, U-87MG, MCF-7 cells, mouse brain, or rat heart tissues .

• Verification: A specific band for EGLN2 should be detected at approximately 43-48 kDa, as demonstrated in MDA-MB-231 human breast cancer cell line lysates .

• Reduction conditions: Western blot experiments should be conducted under reducing conditions using Immunoblot Buffer Group 8 for optimal results .

• Secondary antibody: For sheep-derived primary antibodies, use HRP-conjugated Anti-Sheep IgG Secondary Antibody (such as HAF016) .

What are the key considerations for EGLN2 immunoprecipitation experiments?

For successful immunoprecipitation of EGLN2:

• Antibody amount: Use 0.5-4 μg of antibody for immunoprecipitation from 200-400 μg of whole cell extracts .

• Cross-linking considerations: Due to EGLN2's interaction with multiple proteins (NRF1, PGC1α, FOXO3a), consider using reversible cross-linking approaches to capture transient interactions.

• Buffer compatibility: Choose buffers that maintain protein-protein interactions without disrupting the antibody's binding capacity.

• Nuclear fraction enrichment: Since EGLN2 is enriched in nuclear and chromatin-bound fractions, particularly under hypoxia, consider using subcellular protein fractionation kits when investigating EGLN2's nuclear interactions .

• Verification: Confirm successful immunoprecipitation by Western blot analysis of the immunoprecipitated material.

How can ChIP-seq experiments with EGLN2 antibodies be optimized?

Based on published research using EGLN2 ChIP-seq:

• Hypoxia considerations: EGLN2 shows stronger chromatin binding under hypoxic conditions, with enrichment in nuclear and chromatin-bound fractions . Consider performing parallel experiments under both normoxic and hypoxic conditions.

• Cross-linking protocol: Use formaldehyde (1% final concentration) for 10 minutes at room temperature for effective cross-linking.

• Sonication parameters: Optimize sonication to generate DNA fragments between 200-500 bp.

• Binding partners analysis: Consider dual ChIP approaches to investigate EGLN2's interaction with NRF1 and PGC1α on chromatin, as these interactions are documented .

• Motif analysis: When analyzing ChIP-seq data, look for enrichment of NRF1 motifs in promoters of EGLN2-regulated genes .

• Controls: Include input DNA, IgG controls, and where possible, EGLN2-depleted cells as negative controls.

How can researchers distinguish between EGLN2 and other EglN family members?

To ensure specificity when studying EGLN2 versus EglN1 or EglN3:

• Antibody epitope selection: Choose antibodies targeting unique regions that don't share sequence homology with other family members.

• Knockdown validation: Validate antibody specificity using siRNA targeting specifically EGLN2, EglN1, or EglN3 to confirm signal reduction only upon targeting the intended protein.

• Functional verification: Different EglN family members have distinct functions; for instance, depletion of EGLN2, but not EglN1 or EglN3, decreases mitochondrial respiration in breast cancer cells .

• Molecular weight differentiation: Carefully distinguish between the molecular weights of different family members on Western blots.

• Expression pattern analysis: Consider the differential expression patterns of EglN family members across tissues and cell types.

What approaches can detect post-translational modifications of EGLN2?

For studying EGLN2 post-translational modifications:

• Phosphorylation sites: Research has identified phosphorylation at Thr405 and adjacent Ser401 sites near the C-terminal of EGLN2 . Use phospho-specific antibodies or mass spectrometry approaches.

• Immunoprecipitation followed by mass spectrometry: Purify HA-tagged EGLN2 from cells and examine potential phosphorylation sites by mass spectrometry .

• Mutant analysis: Compare wild-type EGLN2 with mutants (S401A, T405A, ST-AA) to examine the role of phosphorylation sites on protein stability and function .

• Deletion mutants: Consider using deletion mutants (∧TPT or ∧SQPPTPT) that contain specific serine or threonine residues to study their function .

• Hydroxylation assays: For studying EGLN2's hydroxylase activity on substrates, consider in vitro hydroxylation assays that measure the release of CO₂ resulting from the decarboxylation of α-KG .

How can EGLN2 antibodies be used to study cancer mechanisms?

EGLN2 has significant implications in cancer research:

What are the methodological considerations for studying EGLN2 in hypoxic conditions?

When investigating EGLN2 under hypoxia:

• Timing considerations: EGLN2 translocates to the nucleus and shows enrichment in chromatin-bound fractions upon exposure to hypoxia . Consider time-course experiments to capture dynamic changes.

• Subcellular fractionation: Use subcellular protein fractionation kits to separate cytoplasmic, nuclear, and chromatin-bound fractions when comparing normoxic versus hypoxic conditions .

• HIF1/2α-independent functions: Many EGLN2 functions in regulating mitochondrial function occur independently of HIF1/2α . Design experiments that can distinguish between HIF-dependent and HIF-independent mechanisms.

• Transcriptional profiling: EGLN2 shows more robust effects on transcriptional activation under hypoxia compared to normoxia . Consider RNA-seq or microarray analysis to capture these differences.

• Binding partner dynamics: The interaction between EGLN2, NRF1, and PGC1α on chromatin shows stronger binding patterns under hypoxia . Use appropriate co-immunoprecipitation protocols for hypoxic samples.

How can researchers validate EGLN2 antibody specificity?

To ensure antibody specificity:

• Knockout/knockdown validation: Test antibodies in cells where EGLN2 has been depleted via siRNA, shRNA, or CRISPR-Cas9 approaches. Several independent hairpins against EGLN2 have been used to validate antibody specificity .

• Rescue experiments: Confirm specificity by restoring EGLN2 expression with shRNA-resistant EGLN2 constructs .

• Cross-reactivity assessment: Test for potential cross-reactivity with other EglN family members (EglN1, EglN3).

• Positive controls: Include lysates from cells known to express EGLN2 (HeLa, A-549, HepG2, U-87MG, MCF-7, mouse brain, rat heart) .

• Western blot band patterns: A specific band for EGLN2 should be detected at approximately 43-48 kDa .

What are common causes of inconsistent results when using EGLN2 antibodies?

Common issues and solutions include:

• Storage and handling: Aliquot antibodies and store at -20°C to avoid repeated freeze/thaw cycles that can reduce activity .

• Buffer compatibility: Ensure that sample buffers are compatible with the antibody's performance (PBS, pH 7.3, containing 0.02% sodium azide, 50% glycerol is recommended for storage) .

• Protein degradation: Use protease inhibitors during sample preparation to prevent EGLN2 degradation.

• Post-translational modifications: Consider that phosphorylation at Thr405 and Ser401 may affect antibody recognition and protein stability .

• Subcellular localization variations: EGLN2 localizes in both cytoplasm and nucleus, with enrichment in nuclear fractions under hypoxia . Inconsistent results may occur if proper fractionation is not performed.

• Hydroxylation activity: EGLN2's activity as a prolyl hydroxylase may be affected by oxygen levels, potentially influencing antibody recognition of native conformations.