FEN1 Antibody, Biotin conjugated

What is FEN1 and why is it significant in molecular biology research?

FEN1 (Flap structure-specific endonuclease 1) is a multifunctional nuclease that plays critical roles in DNA replication and repair. It possesses both 5'-flap endonuclease and 5'-3' exonuclease activities essential for:

• Processing Okazaki fragments during DNA replication

• Long-patch base excision repair (LP-BER)

• Prevention of genomic instability by cleaving 5'-overhanging flap structures

FEN1 is particularly significant because it acts as a genome stabilization factor that prevents DNA structures from forming duplications and deletions. Its dysregulation has been implicated in various cancers, with overexpression documented in testis, lung, and brain tumors .

What are the advantages of biotin-conjugated FEN1 antibodies over non-conjugated versions?

Biotin-conjugated FEN1 antibodies offer several methodological advantages:

• Enhanced detection sensitivity: The biotin-avidin/streptavidin system provides signal amplification with a dissociation constant of approximately 10^-15 M

• Versatile detection options: Compatible with various secondary detection systems including HRP, fluorescent, or gold-conjugated streptavidin

• Reduced background: Minimizes non-specific binding compared to secondary antibody systems

• Stable conjugation: Biotin remains functional under harsh experimental conditions

• Compatibility with multiplexing: Can be combined with other differently-labeled antibodies for co-localization studies

These properties make biotin-conjugated FEN1 antibodies particularly valuable for detecting low-abundance FEN1 in complex cellular environments or when examining FEN1's post-translational modifications.

What are the optimal buffer and storage conditions for maintaining biotin-conjugated FEN1 antibody activity?

Based on manufacturer specifications, biotin-conjugated FEN1 antibodies require careful handling:

For maximum retention of activity, it's advisable to prepare working dilutions immediately before use and avoid more than 3 freeze-thaw cycles of the stock solution .

What are the recommended experimental conditions for using biotin-conjugated FEN1 antibodies in various applications?

Different applications require specific optimization:

When using high salt extraction methods for chromatin-bound proteins like FEN1, the addition of 0.05% Tween-20 to blocking solutions is recommended to reduce background in Western blot applications .

How can biotin-conjugated FEN1 antibodies be used to study FEN1's post-translational modifications?

FEN1 undergoes multiple post-translational modifications that regulate its functions. Biotin-conjugated FEN1 antibodies can be used to study these modifications through:

• Sequential immunoprecipitation: First IP with biotin-conjugated FEN1 antibody using streptavidin beads, followed by immunoblotting with antibodies against specific modifications:

  • Phosphorylation (primarily at S187)

  • SUMOylation (SUMO-1, SUMO-3 at K354, K366, K367, K369, K375)

  • Methylation (arginine methylation, primarily at R192)

  • Ubiquitination (primarily at K354)

• Co-localization studies: Using biotin-conjugated FEN1 antibodies with antibodies against modification-specific proteins like PRMT5 (methylation), UBE1/UBE2M/PRP19 (ubiquitination), or SUMO ligases .

This methodology has revealed that FEN1 phosphorylation stimulates its SUMOylation, which in turn promotes its ubiquitination and subsequent degradation via the proteasome pathway .

What role does SUMO-1 modification play in FEN1 function and how can this be experimentally demonstrated?

SUMO-1 modification of FEN1 serves as a molecular switch that directs FEN1 from DNA replication to DNA repair functions:

• SUMO-1 modification promotes FEN1 interaction with the Rad9-Rad1-Hus1 complex rather than PCNA

• This modification occurs in response to DNA damage and replication fork stalling

• Key SUMO-1 modification sites include K366, K367, K369, and K375

Experimental demonstration using biotin-conjugated FEN1 antibodies:

• Pull-down assays: Use biotin-conjugated FEN1 antibody with streptavidin beads after cells are treated with DNA damaging agents

• Western blotting: Probe with anti-SUMO-1 antibodies

• Co-immunoprecipitation: Assess interaction with Rad9-Rad1-Hus1 complex versus PCNA

• Site-directed mutagenesis: Create FEN1 mutants (e.g., 4KR mutant) to abolish SUMO-1 modification sites

• Functional assays: Measure DNA repair efficiency in cells expressing wild-type versus mutant FEN1

Results from such experiments have shown that the 4KR FEN1 mutation (K366R, K367R, K369R, K375R) reduces SUMO-1-FEN1 levels in cells under both normal conditions and exposure to DNA-damaging agents .

What are common technical challenges when using biotin-conjugated FEN1 antibodies and how can they be overcome?

For nuclear extraction specifically, FEN1 often requires high salt extraction methods as it is tightly associated with chromatin during DNA replication and repair processes .

How can researchers validate the specificity and sensitivity of biotin-conjugated FEN1 antibodies?

A comprehensive validation strategy should include:

• Positive controls:

  • Use recombinant FEN1 protein at known concentrations

  • Test in cell lines known to express FEN1 (HeLa, NIH/3T3)

• Negative controls:

  • FEN1 knockdown or knockout cell lines

  • Blocking peptide competition assays

  • Secondary-only controls to assess non-specific binding

• Specificity tests:

  • Western blot should detect a band at ~45-48 kDa (observed molecular weight)

  • Immunoprecipitation followed by mass spectrometry

  • Cross-adsorption against related proteins (e.g., other nucleases)

• Sensitivity assessment:

  • Serial dilution of recombinant FEN1 protein

  • Comparison with other validated FEN1 antibodies

  • Limit of detection calculation in specific applications

When validating across applications, researchers should note that FEN1 antibodies may perform differently in fixed versus non-fixed samples due to epitope accessibility changes.

How can biotin-conjugated FEN1 antibodies be employed to study the role of FEN1 in cancer progression?

FEN1 is overexpressed in various cancers, making it a valuable target for cancer research:

• Tissue microarray analysis:

  • Biotin-conjugated FEN1 antibodies enable quantitative assessment of FEN1 expression across cancer stages

  • Data show significant overexpression in 19/25 testicular tumors and 4/4 lung cancer samples

• Chemosensitivity studies:

  • FEN1 levels affect sensitivity to alkylating agents and cisplatin

  • Biotin-conjugated antibodies can track FEN1 levels before and after treatment

  • siRNA down-regulation of FEN1 increases sensitivity to methylating agents and cisplatin in glioma cells

• Mutation analysis pipelines:

  • FEN1 mutations (e.g., L209P) detected in cancer can be studied

  • Pull-down with biotin-conjugated antibodies followed by mass spectrometry can identify altered interaction partners

  • The L209P mutation reduces FEN, EXO and GEN activities of FEN1 while maintaining DNA binding affinity

• Cell cycle-specific regulation:

  • Track FEN1 levels and modifications across cell cycle phases

  • Biotin-conjugated antibodies facilitate flow cytometry analysis when paired with cell cycle markers

What methodological approaches can be used to investigate FEN1's protein-protein interactions using biotin-conjugated antibodies?

Biotin-conjugated FEN1 antibodies enable several advanced interaction study methodologies:

• Sequential Co-immunoprecipitation:

  • First pull-down with biotin-conjugated FEN1 antibody

  • Elution under native conditions

  • Second immunoprecipitation with antibodies against suspected interaction partners

  • This approach revealed that SUMO3-conjugated FEN1 interacts more strongly with the E3 ubiquitin ligase PRP19

• Proximity Ligation Assay (PLA):

  • Use biotin-conjugated FEN1 antibody with antibody against partner protein

  • Detection with oligonucleotide-linked streptavidin

  • Rolling circle amplification produces detectable spots where proteins interact

  • Allows quantitative spatial analysis of interactions in situ

• ChIP-re-ChIP studies:

  • First ChIP with biotin-conjugated FEN1 antibody

  • Second ChIP with antibodies against chromatin-associated partners

  • Identifies genomic regions where FEN1 and partners co-localize

  • Critical for understanding FEN1's role in replication and repair complexes

• BioID or APEX proximity labeling:

  • Express FEN1 fused to BioID or APEX

  • Use biotin-conjugated FEN1 antibodies to confirm expression

  • Identify biotinylated proximity partners by mass spectrometry

  • This approach has identified interaction with UBE1, UBE2M, PRP19 and UBQLN4

These methods have collectively revealed FEN1's dynamic interactions with PCNA during normal replication and with the Rad9-Rad1-Hus1 complex during DNA damage response .