uhrf1 Antibody

What is UHRF1 and why is it significant in molecular biology research?

UHRF1, also known as ICBP90, NP95, or RNF106, functions as a crucial transcription and cell cycle regulator belonging to the RING-finger type E3 ubiquitin ligase subfamily. It contains a distinctive structure featuring one PHD-type zinc finger, a ubiquitin-like domain, two RING-type zinc fingers, and one YDG/SRA domain, collectively facilitating its function as an E3 ubiquitin-protein ligase. UHRF1 is primarily localized in the nucleus where it plays a vital role in mediating ubiquitination processes essential for protein degradation and cellular proliferation. Its direct interaction with Dnmt1 (a maintenance DNA methyltransferase) ensures stable association with chromatin, critical for regulating gene expression and epigenetic inheritance. The protein's overexpression in cancer cells highlights its potential role in carcinogenesis, making it a significant target for cancer biology research and therapeutic intervention development .

What types of UHRF1 antibodies are available for research applications?

Researchers can access several types of UHRF1 antibodies, including monoclonal antibodies like mouse monoclonal IgG1 kappa light chain antibodies (e.g., H-8) that detect UHRF1 protein across multiple species (mouse, rat, and human) . Additionally, polyclonal antibodies such as rabbit polyclonal IgG antibodies are available that demonstrate reactivity with human samples and potentially with mouse samples . These antibodies come in both non-conjugated forms and various conjugated forms, including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates, offering researchers flexibility in experimental design .

What are the typical applications for UHRF1 antibodies in laboratory research?

UHRF1 antibodies can be utilized across numerous research applications, including:

• Western blotting (WB) for protein expression analysis

• Immunoprecipitation (IP) for protein complex isolation

• Immunofluorescence (IF) and immunocytochemistry (ICC) for subcellular localization studies

• Immunohistochemistry (IHC) for tissue expression patterns

• Flow cytometry for intracellular protein analysis

• Enzyme-linked immunosorbent assay (ELISA) for quantitative measurements

• Co-immunoprecipitation (Co-IP) for protein-protein interaction studies

• RNA immunoprecipitation (RIP) for RNA-protein interaction analysis

This versatility makes UHRF1 antibodies valuable tools for investigating multiple aspects of UHRF1 biology across different experimental contexts.

How should researchers optimize western blotting protocols for UHRF1 detection?

For optimal western blotting of UHRF1, researchers should consider the following methodological approach:

• Sample preparation: Extract proteins using RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors (especially when studying phosphorylated forms of UHRF1).

• Gel electrophoresis: Use 8-10% SDS-PAGE gels as UHRF1 has an expected molecular weight of approximately 90-100 kDa (observed molecular weight: 91-100 kDa) .

• Transfer conditions: Employ semi-dry or wet transfer methods with methanol-containing transfer buffer for efficient protein transfer.

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute UHRF1 antibodies according to manufacturer recommendations (typically 1:500-1:2000) and incubate overnight at 4°C.

• Detection: Use species-appropriate HRP-conjugated secondary antibodies followed by enhanced chemiluminescence detection.

When analyzing results, researchers should expect to observe UHRF1 bands between 91-100 kDa . Some antibodies might detect phosphorylated forms of UHRF1, potentially appearing as higher molecular weight bands, especially when cells are treated with DNA damaging agents or arrested in specific cell cycle phases .

What methodological considerations are important when using UHRF1 antibodies for immunoprecipitation studies?

When conducting immunoprecipitation with UHRF1 antibodies, researchers should implement the following methodological approaches:

• Lysis conditions: Use mild lysis buffers (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 5 mM EDTA with protease and phosphatase inhibitors) to preserve protein-protein interactions.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody binding: Incubate cell lysates with UHRF1 antibodies (2-5 μg per 500 μg of protein) overnight at 4°C with gentle rotation.

• Precipitation: Add protein A/G beads for 2-4 hours at 4°C.

• Washing: Perform stringent washing (at least 4-5 washes) to remove non-specific interactions.

• Elution: Elute proteins with SDS sample buffer by heating at 95°C for 5 minutes.

This approach has successfully identified UHRF1-interacting proteins such as HDAC1, PCNA, USP7, and USP11 in previous studies . Researchers should note that interactions may be enhanced following DNA damage, as demonstrated with XLF interaction , and therefore consider treatment conditions that reflect their research question. For studying ubiquitination of UHRF1 or its targets, researchers should include deubiquitinase inhibitors (e.g., N-ethylmaleimide) in lysis buffers and consider using tagged ubiquitin constructs for more specific detection .

How can researchers effectively implement immunofluorescence studies using UHRF1 antibodies?

For successful immunofluorescence experiments with UHRF1 antibodies, researchers should follow these methodological guidelines:

• Cell preparation: Culture cells on coverslips or chamber slides at 50-70% confluency to allow clear visualization of nuclear structures.

• Fixation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature to preserve protein localization and epitope accessibility.

• Permeabilization: Permeabilize with 0.2% Triton X-100 for 10 minutes to allow antibody access to nuclear UHRF1.

• Blocking: Block with 5% normal serum from the species of the secondary antibody for 1 hour to reduce background.

• Primary antibody incubation: Dilute UHRF1 antibodies according to manufacturer recommendations (typically 1:50-1:200) and incubate overnight at 4°C.

• Secondary antibody incubation: Use fluorescently labeled secondary antibodies at 1:500-1:1000 dilution for 1 hour at room temperature.

• Counterstaining: Counterstain nuclei with DAPI and mount with anti-fade mounting medium.

When analyzing results, expect to observe primarily nuclear localization of UHRF1, with possible enrichment in specific nuclear structures during certain cell cycle phases. For co-localization studies, such as those examining UHRF1 and DNA damage markers, laser micro-irradiation experiments have shown that both exogenous and endogenous UHRF1 co-localize with DNA repair factors like XLF at sites of DNA damage . This approach provides valuable insights into UHRF1's dynamic localization during cellular processes.

How can UHRF1 antibodies be utilized to study DNA damage response mechanisms?

UHRF1 antibodies serve as powerful tools for investigating DNA damage response mechanisms through several methodological approaches:

• DNA damage recruitment studies: After inducing DNA double-strand breaks (using laser micro-irradiation, ionizing radiation, or chemical agents like etoposide), researchers can employ UHRF1 antibodies in immunofluorescence assays to monitor UHRF1 recruitment to damage sites. Evidence indicates that both exogenously expressed and endogenous UHRF1 co-localize with DNA repair factors at laser-induced DNA damage stripes .

• Protein interaction analyses: Researchers can utilize co-immunoprecipitation with UHRF1 antibodies to identify damage-induced protein interactions. For instance, studies have revealed that UHRF1 interaction with the non-homologous end joining factor XLF is enhanced following DNA damage . This experiment requires:

  • Treating cells with DNA-damaging agents (e.g., 10 Gy irradiation)

  • Performing immunoprecipitation with UHRF1 antibodies

  • Blotting for interaction partners such as XLF

• Post-translational modification assessment: UHRF1 antibodies can be used to investigate how DNA damage affects UHRF1 modifications, including ubiquitination status. Research has shown that UHRF1 catalyzes K63-linked (rather than K48-linked) polyubiquitination of DNA repair factors like XLF following DNA damage . To study this:

  • Immunoprecipitate proteins of interest after damage induction

  • Probe with ubiquitin antibodies (such as FK2)

  • Compare results between control and UHRF1-depleted cells

• Functional studies: By combining UHRF1 antibodies with CRISPR-mediated UHRF1 depletion, researchers can assess how UHRF1 loss affects ubiquitination of repair factors and subsequent DNA damage repair kinetics .

What methods can be employed to study UHRF1 phosphorylation states using phospho-specific antibodies?

To investigate UHRF1 phosphorylation states, researchers should implement the following methodological approaches:

• Phospho-specific antibody generation and validation:

  • Generate antibodies against specific phosphorylation sites, such as Serine 652 (S652ph)

  • Validate specificity using peptide competition assays with phosphorylated and non-phosphorylated peptides

  • Confirm reactivity with wild-type but not phospho-site mutant (e.g., S652A) UHRF1 proteins

  • Verify antibody specificity through UHRF1 knockdown experiments

• Cell cycle synchronization for phosphorylation studies:

  • Synchronize cells using methods like double thymidine block (G1/S phase arrest) or thymidine-nocodazole treatment (M phase arrest)

  • Confirm synchronization efficiency using flow cytometry

  • Prepare extracts from synchronized cells and analyze UHRF1 phosphorylation status

• Investigation of kinase-dependent phosphorylation:

  • Treat cells with specific kinase inhibitors

  • Compare phosphorylation levels before and after treatment

  • Use in vitro kinase assays with purified components to confirm direct phosphorylation

• Functional analysis of phosphorylation:

  • Generate phospho-mimetic (S→D or S→E) and phospho-deficient (S→A) mutants

  • Perform rescue experiments in UHRF1-depleted cells

  • Assess the impact on protein interactions (e.g., S652 phosphorylation potentially affects USP7 interaction)

Research has demonstrated that UHRF1 is phosphorylated during M phase, potentially regulating its association with the deubiquitylase USP7, which has implications for UHRF1 stability and function .

How can researchers effectively study UHRF1-mediated ubiquitination processes?

To investigate UHRF1-mediated ubiquitination processes, researchers should implement these methodological approaches:

• In vivo ubiquitination assays:

  • Transfect cells with HA-tagged ubiquitin and relevant substrate constructs

  • Treat cells with proteasome inhibitors (e.g., MG132) to prevent degradation of ubiquitinated proteins

  • Immunoprecipitate the substrate of interest

  • Probe with anti-HA antibodies to detect ubiquitination

  • Compare results between wild-type cells and cells with UHRF1 depletion or overexpression

• In vitro ubiquitination assays:

  • Purify recombinant His-tagged UHRF1 and substrate proteins (e.g., XLF)

  • Combine with E1, E2 enzymes, ATP, and ubiquitin

  • Incubate at 37°C for 1-2 hours

  • Analyze ubiquitination by Western blotting with substrate-specific or ubiquitin-specific antibodies

• Ubiquitin linkage-specific analysis:

  • Use linkage-specific antibodies (e.g., K48-specific or K63-specific) to determine ubiquitin chain types

  • Alternatively, use ubiquitin mutants that contain only specific lysine residues

  • This approach has revealed that UHRF1 catalyzes K63-linked (rather than K48-linked) polyubiquitination of factors like XLF

• Functional consequences of ubiquitination:

  • Assess protein stability through cycloheximide chase experiments

  • Examine protein localization changes following ubiquitination

  • Investigate protein-protein interactions affected by ubiquitination

  • Study recruitment to specific cellular structures (e.g., enhanced recruitment of XLF to DNA damage sites following UHRF1-mediated ubiquitination)

How should researchers address non-specific binding issues when using UHRF1 antibodies?

When encountering non-specific binding with UHRF1 antibodies, researchers should implement the following troubleshooting strategies:

• Antibody validation:

  • Confirm antibody specificity using UHRF1 knockdown or knockout controls

  • Compare results from multiple UHRF1 antibodies targeting different epitopes

  • Include appropriate negative controls (secondary antibody only, isotype controls)

• Western blotting optimization:

  • Increase blocking stringency (5-10% milk/BSA, longer blocking times)

  • Optimize primary antibody dilution (perform titration experiments)

  • Increase washing duration and number of washes

  • Use clean, freshly prepared buffers

  • Consider alternative blocking agents (casein, fish gelatin)

• Immunofluorescence/immunohistochemistry optimization:

  • Implement appropriate antigen retrieval methods

  • Test different fixation protocols (paraformaldehyde vs. methanol)

  • Increase blocking time and concentration

  • Pre-absorb antibodies with cell/tissue lysates

  • Optimize antibody concentrations

• Data interpretation guidance:

  • Focus on bands at the expected molecular weight (91-100 kDa for UHRF1)

  • Be aware that phospho-specific antibodies may detect other high molecular weight bands that are not UHRF1-specific

  • Consider that immunoprecipitation followed by Western blotting may reveal more specific results than direct Western blotting

How can researchers resolve contradictory results when studying UHRF1 protein interactions?

When faced with contradictory results in UHRF1 interaction studies, researchers should implement the following systematic approach:

• Experimental condition variations:

  • Evaluate cell cycle-dependent interactions by synchronizing cells at different cell cycle phases

  • Test interactions under various cellular stresses (DNA damage, metabolic stress)

  • Research indicates that UHRF1 interaction with XLF is enhanced following DNA damage , and cell cycle phase may affect interactions with USP7

• Interaction detection techniques:

  • Compare results from multiple methods (co-IP, proximity ligation assay, FRET)

  • Use reciprocal immunoprecipitation (IP with anti-UHRF1 vs. IP with antibody against the interacting protein)

  • For weak or transient interactions, consider chemical crosslinking before lysis

  • Verify endogenous interactions rather than relying solely on overexpression systems

• Protein domain mapping:

  • Generate and test truncation mutants to identify critical interaction domains

  • Consider how post-translational modifications might affect interactions

  • For instance, serine 652 phosphorylation in UHRF1 may regulate its association with USP7

• Cell type and context considerations:

  • Compare interactions across different cell lines and tissue types

  • Assess interaction dynamics during differentiation or disease progression

  • Consider species-specific differences in interaction patterns

• Technical validation:

  • Confirm antibody specificity for both UHRF1 and the interacting protein

  • Optimize lysis conditions to preserve interactions (test different detergents and salt concentrations)

  • Include appropriate controls (IgG control, lysate input)

By systematically addressing these factors, researchers can resolve contradictory results and gain more accurate insights into the complex interactome of UHRF1 under varying cellular conditions.

What strategies should be employed when interpreting UHRF1 localization patterns in immunofluorescence studies?

When interpreting UHRF1 localization patterns in immunofluorescence studies, researchers should implement these analytical strategies:

• Cell cycle-dependent localization analysis:

  • Synchronize cells at different cell cycle phases or use cell cycle markers

  • Document how UHRF1 localization patterns change throughout the cell cycle

  • UHRF1 is known to be enriched in pericentric heterochromatin during specific cell cycle phases

• Co-localization with functional markers:

  • Perform dual staining with markers for specific nuclear domains:

    • DNA replication foci (PCNA)

    • Heterochromatin (H3K9me3)

    • DNA damage sites (γH2AX, 53BP1)

  • Research has demonstrated that UHRF1 co-localizes with DNA repair factors like XLF at laser-induced DNA damage sites

• Technical considerations for accurate interpretation:

  • Use appropriate controls to determine antibody specificity:

    • UHRF1 knockdown/knockout cells

    • Peptide competition assays

  • Apply deconvolution or super-resolution microscopy for detailed localization studies

  • Compare multiple fixation methods to avoid fixation artifacts

  • Evaluate three-dimensional distribution using Z-stack imaging

• Dynamic localization studies:

  • Consider live-cell imaging with fluorescently tagged UHRF1

  • Implement fluorescence recovery after photobleaching (FRAP) to assess mobility

  • Employ techniques like laser micro-irradiation to study recruitment to specific structures

• Interpretation challenges and solutions:

  • Distinguish true nuclear localization from cytoplasmic background

  • Be aware that overexpression may cause artifacts in localization patterns

  • Consider that fixation conditions may affect epitope accessibility

  • Validate key findings with orthogonal approaches (biochemical fractionation)

How can UHRF1 antibodies be utilized to investigate epigenetic inheritance mechanisms?

UHRF1 antibodies offer powerful approaches for investigating epigenetic inheritance mechanisms through these methodological strategies:

• DNA methylation maintenance analysis:

  • Chromatin immunoprecipitation (ChIP) using UHRF1 antibodies followed by sequencing (ChIP-seq) to map UHRF1 binding sites genome-wide

  • Sequential ChIP (UHRF1 followed by DNMT1) to identify regions where both proteins co-occupy

  • Combined bisulfite restriction analysis (COBRA) or bisulfite sequencing of UHRF1-bound regions to correlate binding with DNA methylation status

  • UHRF1 specifically recognizes and binds hemimethylated DNA at replication forks via its YDG domain and recruits DNMT1 methyltransferase to ensure faithful propagation of DNA methylation patterns through DNA replication

• Histone modification recognition studies:

  • Use UHRF1 antibodies in conjunction with histone modification-specific antibodies to analyze co-localization patterns

  • Perform peptide pulldown assays with modified histone peptides followed by UHRF1 immunoblotting

  • UHRF1's Tudor-like regions and PHD-type zinc fingers specifically recognize histone H3 trimethylated at 'Lys-9' (H3K9me3) and unmethylated at 'Arg-2' (H3R2me0), respectively

• Cell division-coupled epigenetic inheritance:

  • Synchronize cells and collect samples at different cell cycle stages

  • Perform UHRF1 immunoprecipitation followed by mass spectrometry to identify cell cycle-specific interaction partners

  • Use UHRF1 antibodies to track its localization during DNA replication by co-staining with replication markers

• UHRF1 post-translational modification influence on epigenetic function:

  • Utilize phospho-specific UHRF1 antibodies to determine how phosphorylation impacts UHRF1's epigenetic functions

  • Investigate how UHRF1's E3 ubiquitin ligase activity affects histone modifications by combining UHRF1 immunoprecipitation with ubiquitin antibodies

  • Research has shown that M phase phosphorylation of UHRF1 may regulate its stability and function

What methodological approaches can be used to study UHRF1's role in cancer progression?

To investigate UHRF1's role in cancer progression, researchers should implement these methodological approaches:

• Expression analysis across cancer types:

  • Immunohistochemistry with UHRF1 antibodies on tissue microarrays containing normal and cancerous tissues

  • Quantitative analysis of staining intensity and subcellular localization

  • Correlation of UHRF1 expression with clinical parameters and patient outcomes

  • Research has demonstrated UHRF1 overexpression in various cancer cells, highlighting its potential role in carcinogenesis

• Functional studies in cancer models:

  • UHRF1 knockdown/knockout in cancer cell lines using CRISPR-Cas9

  • Phenotypic analysis of proliferation, migration, invasion, and apoptosis

  • Rescue experiments with wild-type or mutant UHRF1

  • In vivo tumor formation studies in xenograft models

• Mechanistic investigations:

  • ChIP-seq analysis to identify cancer-specific UHRF1 binding sites

  • RNA-seq after UHRF1 modulation to identify regulated genes

  • DNA methylation analysis (reduced representation bisulfite sequencing, RRBS) to correlate UHRF1 levels with methylation patterns

  • Protein interaction studies in normal versus cancer cells

• Therapeutic targeting approaches:

  • Screening for small molecule inhibitors of UHRF1

  • Analysis of UHRF1 as a biomarker for treatment response

  • Combination studies with epigenetic drugs (DNMT inhibitors, HDAC inhibitors)

  • Evaluation of synthetic lethality opportunities

• Pathway integration analysis:

  • Investigation of UHRF1's role in specific cancer-associated pathways

  • Study of UHRF1 in relation to metabolic regulation (UHRF1 has been identified as a novel metabolic guardian restricting AMPK activity)

  • Analysis of UHRF1's involvement in cancer stem cell maintenance

How can researchers investigate the relationship between UHRF1 and other epigenetic regulators using antibody-based approaches?

To investigate the relationship between UHRF1 and other epigenetic regulators, researchers should implement these antibody-based methodological approaches:

• Multi-protein complex analysis:

  • Tandem affinity purification using UHRF1 antibodies followed by mass spectrometry

  • Sequential ChIP (ChIP-reChIP) to identify genomic regions co-occupied by UHRF1 and other epigenetic factors

  • Proximity ligation assay (PLA) to visualize and quantify in situ interactions between UHRF1 and other epigenetic regulators

  • Research has identified interactions between UHRF1 and epigenetic regulators such as DNMT1, HDAC1, and deubiquitinases USP7/USP11

• Functional interdependency studies:

  • Depletion of UHRF1 followed by ChIP-seq for other epigenetic regulators

  • Analysis of epigenetic mark changes (DNA methylation, histone modifications) after UHRF1 modulation

  • Rescue experiments combining UHRF1 knockdown with overexpression of other epigenetic factors

• Chromatin state correlation:

  • Combine UHRF1 ChIP-seq with histone modification ChIP-seq

  • Integrate DNA methylation data with UHRF1 binding patterns

  • UHRF1 is known to specifically recognize histone H3 trimethylated at 'Lys-9' (H3K9me3) and unmethylated at 'Arg-2' (H3R2me0) through its tudor-like regions and PHD-type zinc fingers, respectively

• Post-translational regulation network:

  • Use specific antibodies to study how modifications of UHRF1 affect its interaction with other epigenetic regulators

  • Investigate how UHRF1's E3 ligase activity regulates other epigenetic factors

  • Research has shown that UHRF1 prevents excessive DNA methylation by methylation-mediated degradation of itself and DNMT1

• Genome editing approaches:

  • CRISPR-Cas9 domain mutagenesis to disrupt specific UHRF1 interactions

  • Create anchor-away systems to study the consequences of UHRF1 removal from chromatin

  • Generate degron-tagged UHRF1 for rapid protein depletion studies

By integrating these approaches, researchers can comprehensively map the functional relationships between UHRF1 and the broader epigenetic regulatory network, providing insights into fundamental epigenetic mechanisms and potential therapeutic interventions in diseases associated with epigenetic dysregulation.