UHRF1 Monoclonal Antibody

How can researchers validate the specificity of a UHRF1 monoclonal antibody across different experimental systems?

Validation requires a multi-step approach combining knockout controls, cross-reactivity assays, and orthogonal validation methods. For western blotting, lysates from UHRF1 knockout cell lines (e.g., CRISPR-edited HeLa or HEK293T) should show no band at the expected molecular weight (91–100 kDa) . Immunofluorescence (IF) specificity can be confirmed by comparing nuclear localization patterns in wild-type versus knockout cells, as UHRF1 is predominantly nuclear . Cross-reactivity risks are mitigated by testing antibody performance in species beyond humans (e.g., mouse or rat models) . Proteintech’s antibody (21402-1-AP) demonstrates specificity in HuH-7, HEK-293T, and MCF-7 cells via WB, while Santa Cruz’s H-8 antibody is validated in mouse and rat tissues .

What methodological considerations are critical for optimizing UHRF1 antibody dilution in chromatin immunoprecipitation (ChIP) assays?

ChIP-grade UHRF1 antibodies require empirical titration due to chromatin accessibility challenges. A starting dilution of 1:50–1:100 is recommended, with verification via:

• Positive controls: Known UHRF1-binding regions (e.g., TOP2A promoter) .

• Negative controls: IgG isotype and UHRF1 knockout lysates .
  Proteintech’s IF/ICC protocol suggests 1:50–1:500 dilutions, but ChIP may require higher concentrations due to epitope masking . Pre-clearing chromatin lysates with protein A/G beads reduces non-specific binding.

How does UHRF1 monoclonal antibody selection impact DNA methylation studies?

UHRF1’s role in recruiting DNMT1 necessitates antibodies that preserve native protein-DNA interactions. Antibodies targeting the SRA domain (e.g., Abcam’s ab57083) are optimal for studying hemimethylated DNA recognition, while those binding the PHD domain (e.g., Santa Cruz’s H-8) may disrupt histone interaction . Co-IP experiments using UHRF1 antibodies should confirm DNMT1 co-precipitation, as this interaction is essential for methylation maintenance .

How can researchers resolve contradictions in UHRF1’s reported roles in epigenetic silencing versus transcriptional activation?

Discrepancies arise from cell type-specific contexts and post-translational modifications. For example:

• In cancer cells, UHRF1 represses tumor suppressors (e.g., CDKN2A) via H3K9me3 binding .

• During G1/S transition, UHRF1 activates TOP2A by binding unmethylated H3R2 .
  To reconcile these, employ phospho-specific antibodies (e.g., targeting phosphorylation at S/T residues) and time-resolved ChIP-seq to map UHRF1’s dynamic localization. Proteintech’s antibody has been used in studies linking UHRF1 to AMPK-mediated metabolic regulation, illustrating its context-dependent functions .

What experimental strategies identify UHRF1’s non-canonical roles in mitotic spindle regulation?

Recent work reveals UHRF1’s interaction with EG5 kinesin and TPX2 during metaphase . To study this:

• Use mitotic synchronization (e.g., nocodazole block) followed by immunofluorescence with UHRF1 antibodies.

• Validate spindle localization via co-staining with α-tubulin and EG5 .

• Perform Ubiquitination assays to test if UHRF1 catalyzes K63-linked polyubiquitination of EG5, as reported in HeLa cells .
  Santa Cruz’s H-8 antibody detects UHRF1 in mitotic spindles, making it suitable for these assays .

How do researchers address variability in UHRF1 antibody performance across cancer subtypes?

Variability stems from isoform expression and post-translational modifications. For example:

• Isoform 2 (NP_001261479) lacks the TTD domain, altering chromatin binding .

• Phosphorylation at Ser 674 modulates DNMT1 recruitment .
  Solutions include:

• Isoform-specific antibodies: Proteintech’s 21402-1-AP detects all isoforms, while custom antibodies targeting isoform-unique regions improve specificity.

• Phospho-proteomics: Combine IP with mass spectrometry to map modification states .

What controls are essential for UHRF1 co-immunoprecipitation (Co-IP) experiments?

• Negative controls: IgG isotype, UHRF1 knockout lysates.

• Input normalization: 10% of lysate reserved for input blotting.

• Competition assays: Pre-incubate antibody with immunogen peptide to block binding .
  Abcam’s ab57083 has been used in Co-IP studies with DNMT1, requiring 0.5–4.0 µg antibody per mg lysate .

How can researchers optimize UHRF1 antibody storage to prevent epitope degradation?

• Aliquoting: Avoid repeated freeze-thaw cycles; 20 µL aliquots in PBS with 50% glycerol (Proteintech’s formulation) stabilize reactivity .

• Long-term storage: -80°C for >2 years; -20°C for routine use .

What bioinformatics tools complement UHRF1 antibody-based studies in epigenomics?

• ChIP-Seq: Peak calling with MACS2, annotated against ENCODE UHRF1 datasets.

• Motif analysis: MEME Suite to identify UHRF1-binding motifs (e.g., 5’-CCAAT-3’ inverted repeats) .

• Pathway enrichment: DAVID or Metascape for linking targets to pathways like p53 signaling or DNA repair .

How are conflicting WB results for UHRF1 molecular weight resolved?

UHRF1 migrates at 91–100 kDa due to phosphorylation and ubiquitination . Strategies include:

• Phosphatase treatment: Pre-incubate lysates with λ-phosphatase.

• Two-dimensional gel electrophoresis: Separate isoforms by pI and molecular weight.

• Alternative antibodies: Compare results from N-terminal (Proteintech) vs. C-terminal (Santa Cruz H-8) antibodies .

What novel roles for UHRF1 are revealed by CRISPR-Cas9 synergy?

CRISPR knockout screens identify UHRF1 as a synthetic lethal target in DNMT1-deficient cancers. Antibodies like ab57083 validate UHRF1 degradation via PROTACs, showing >70% reduction in viability in DNMT1-/- models .