UBTF Antibody

What is UBTF and what cellular functions does it regulate?

UBTF is a nucleolar protein primarily associated with active transcription of ribosomal DNA (rDNA) and plays an essential role in nucleolar formation. It is necessary for embryonic development past the morula stage, with UBTF knockout cells displaying nucleolar disassembly, abnormal heterochromatin distribution on active rDNA, and loss of rRNA synthesis .

Beyond its canonical role in rRNA biogenesis, UBTF has recently gained increased interest in hematological malignancies, including acute myeloid leukemia (AML) and B-cell acute lymphoblastic leukemia . Recurrent tandem duplications in UBTF (UBTF-TDs), particularly in exon 13, have emerged as a major subtype-defining genomic alteration in pediatric AML that is associated with poor prognosis .

What types of UBTF antibodies are available for research applications?

Several types of UBTF antibodies are available for research applications:

The choice of antibody depends on the specific research application and whether you're interested in detecting total UBTF protein or a specific phosphorylated form .

What is the recommended protocol for UBTF antibody storage to maintain activity?

For optimal preservation of UBTF antibody activity:

• Store antibodies at -20°C or lower

• Aliquot antibodies to avoid repeated freezing and thawing cycles

• Most UBTF antibodies are stored in buffers such as 1x PBS (pH 7.4), sometimes with additives like sodium azide (0.05%) and glycerol (40%)

• Before use, thaw aliquots completely and keep on ice while working

• Avoid exposing antibodies to room temperature for extended periods

Poor storage conditions can lead to antibody degradation, resulting in reduced binding specificity and increased background in experimental applications .

How should I optimize Western blot protocols for UBTF detection?

For optimal UBTF detection by Western blotting:

• Sample preparation:

  • For nuclear proteins like UBTF, use nuclear extraction protocols or subcellular fractionation

  • Normalize samples by protein concentration before loading

• Gel selection:

  • Use 6% SDS-PAGE gels for optimal separation of UBTF (~89 kDa)

• Transfer and blocking:

  • Transfer time may need to be extended for larger proteins like UBTF

  • Use 5% non-fat dry milk or BSA in TBST for blocking

• Antibody dilution:

  • Primary antibody dilutions vary by manufacturer (typically 1:500-1:1000)

  • For the UBTF monoclonal antibody 6B6, experimental determination of optimal dilution is recommended

• Detection and validation:

  • Include positive controls such as HeLa S3 nuclear extract

  • For phospho-specific UBTF antibodies, include both phosphorylated and non-phosphorylated controls

The research by Vlaming et al. demonstrated successful UBTF gene silencing validation using Western blotting after normalization by protein concentration, showing clear differences between control and knockdown samples .

What controls should I include when using UBTF antibodies for immunohistochemistry or immunofluorescence?

When performing immunohistochemistry (IHC) or immunofluorescence (IF) with UBTF antibodies, include the following controls:

Positive controls:

• Known UBTF-expressing tissues/cells (e.g., testis for IHC, HeLa cells for IF)

• Multiple antibody concentrations to determine optimal staining (recommended starting points: 3 μg/ml for IHC-P, 10 μg/ml for IF)

Negative controls:

• Omission of primary antibody

• Isotype control antibody (e.g., mouse IgG2a kappa for monoclonal antibodies)

• UBTF-depleted samples (using shRNA knockdown)

Specificity controls:

• Subcellular localization validation - UBTF should show primarily nucleolar localization

• Use nucleolar markers like fibrillarin as co-staining to confirm proper nucleolar detection

• For phospho-specific antibodies, include dephosphorylated samples as controls

In the study by Vlaming et al., researchers used antibody against fibrillarin as a nucleolar marker and antibody against α-tubulin as a cytosolic marker to confirm successful subcellular fractionation when analyzing UBTF localization .

How do I validate the specificity of a UBTF antibody for my experimental system?

To validate UBTF antibody specificity:

• Western blot analysis:

  • Confirm a single band of expected molecular weight (~89 kDa)

  • Compare with recombinant UBTF protein as a positive control

  • Test in cell lines with known UBTF expression (e.g., HeLa, NIH/3T3)

• Knockdown/knockout validation:

  • Perform shRNA-mediated knockdown of UBTF

  • Compare antibody signal between control and UBTF-depleted samples

  • A specific antibody should show reduced signal in knockdown samples

• Immunoprecipitation:

  • Perform IP with the UBTF antibody followed by mass spectrometry or Western blot

  • Confirm that UBTF is the predominant protein pulled down

• Peptide competition:

  • Pre-incubate antibody with the immunizing peptide

  • A specific antibody should show reduced or abolished staining

• Cross-reactivity testing:

  • Test the antibody against samples from different species if cross-reactivity is claimed

  • Verify antibody performance in your specific experimental system

In Vlaming et al.'s research, they confirmed UBTF knockdown efficiency using Western blotting after transducing cells with shRNA vectors, demonstrating that three out of four shRNA constructs successfully reduced UBTF protein levels .

How can UBTF antibodies be used to investigate the role of UBTF tandem duplications (UBTF-TDs) in leukemia?

UBTF-TDs have emerged as significant genomic alterations in pediatric and adult AML with poor prognosis. For investigating UBTF-TDs:

• Detection and characterization:

  • Use PCR with 6-FAM labeled primers covering exon 13 followed by high-resolution fragment analysis to screen for UBTF-TDs

  • Sequence PCR products to confirm tandem duplications

  • Use targeted NGS-based resequencing to identify co-mutations in UBTF-mutant patients

• Functional studies with antibodies:

  • Compare wild-type UBTF and UBTF-TD localization using immunofluorescence

  • Perform ChIP-seq with UBTF antibodies to compare genomic occupancy patterns

  • Research by Barajas et al. found that UBTF-TD protein maintained genomic occupancy at rDNA loci while also occupying genomic targets like HOXA/HOXB gene clusters and MEIS1

• Protein interaction studies:

  • Use co-immunoprecipitation with UBTF antibodies to identify differential protein interactions between wild-type UBTF and UBTF-TD

  • UBTF-TD co-occupies key genomic loci with KMT2A and menin, suggesting therapeutic potential for menin inhibitors

• Therapeutic response monitoring:

  • Use UBTF antibodies to track protein localization and levels during treatment with menin inhibitors like SNDX-5613

  • Monitor UBTF-TD-mediated transcriptional signatures before and after treatment

Research has demonstrated that UBTF-TD is a gain-of-function alteration resulting in mislocalization to genomic loci dysregulated in UBTF-TD leukemias, and that UBTF-TD AMLs are sensitive to menin inhibition .

What methodologies can be used to distinguish between UBTF1 and UBTF2 isoforms in experimental systems?

UBTF has two major splice variants, UBTF1 and UBTF2, with different functional properties:

• Isoform-specific detection:

  • Western blotting using SDS-PAGE with 6-8% gels for optimal separation of UBTF1 (~97 kDa) and UBTF2 (~89 kDa)

  • If using tagged constructs, anti-tag antibodies (e.g., anti-HA) can distinguish overexpressed UBTF1 and UBTF2 from endogenous UBTF

• Subcellular fractionation:

  • Separate cells into cytoplasmic, soluble nuclear, and chromatin-bound nuclear fractions

  • Analyze distribution patterns of UBTF1 vs UBTF2 across fractions

  • Use markers like fibrillarin (nucleolar) and α-tubulin (cytosolic) to confirm successful fractionation

• ChIP-seq analysis:

  • Perform chromatin immunoprecipitation with UBTF antibodies followed by sequencing

  • Compare genomic binding profiles of UBTF1 and UBTF2

  • Studies have identified differential binding patterns, with approximately 46-52% of UBTF1/2 binding occurring at exon/intron regions of RefSeq genes

• qChIP for target validation:

  • Use quantitative ChIP to validate binding at specific targets

  • Compare enrichment at genes like Asf1a, Smarca5, Dnmt3a, Dut, Myc, and Smc4

Research by Sanij et al. identified differential roles for UBTF1/2 in maintaining genome stability, with gene ontology analysis revealing UBTF1/2-bound regions involved in regulating cell cycle checkpoints, DNA damage responses, and ATR/ATM-regulated DNA damage responses .

How can I effectively use UBTF antibodies to study the role of UBTF in ribosomal DNA regulation and nucleolar function?

For studying UBTF's role in rDNA regulation and nucleolar function:

• ChIP-seq for rDNA occupancy:

  • Use UBTF antibodies for chromatin immunoprecipitation followed by sequencing

  • Compare rDNA occupancy patterns between normal and disease states

  • Research shows UBTF binding at rDNA is a shared feature of both wild-type UBTF and UBTF-TD

• Nucleolar organization analysis:

  • Use immunofluorescence with UBTF antibodies to visualize nucleolar structures

  • Co-stain with other nucleolar markers (e.g., fibrillarin)

  • Analyze changes in nucleolar morphology and UBTF distribution under various conditions

• rRNA synthesis assays:

  • Combine UBTF knockdown or overexpression with rRNA synthesis measurements

  • Use UBTF antibodies to confirm protein levels and localization

  • Both wild-type UBTF and UBTF-TD appear to decrease inactive rDNA sites compared to controls

• Protein degradation systems:

  • Use FKBP12F36V::HA-UBTF fusion constructs with dTAG-13 for rapid degradation

  • Monitor effects on rDNA activity and nucleolar structure

  • Research shows degradation of UBTF-TD results in decreased proliferation and increased myeloid differentiation

• Cell cycle analysis:

  • Study UBTF phosphorylation changes throughout the cell cycle using phospho-specific antibodies

  • UBTF phosphorylation at Ser484 can be detected with specific antibodies

  • Ubtf1/2 depletion has been shown to cause delay in G1-S progression

Studies have demonstrated that UBTF is necessary for nucleolar formation and rRNA synthesis, with knockout cells displaying nucleolar disassembly and abnormal heterochromatin distribution on active rDNA .

What are the best approaches for using UBTF antibodies in site-specific ubiquitination studies?

While studying site-specific ubiquitination of UBTF is challenging, several approaches can be effective:

• Development of site-specific ubiquitin antibodies:

  • Chemical synthesis methods for antigen preparation using full-length ubiquitin and derivatives

  • Application of chemical ligation technologies to synthesize well-defined Ub-modified polypeptides

  • Use of either native isopeptide linkage via thiolysine-mediated ligation or stable bond using click chemistry

• Screening strategy for site-specific antibodies:

  • Design and synthesize non-hydrolyzable Ub-peptide conjugates for immunization

  • Create extended native iso-peptide linked Ub-peptide conjugates for screening

  • Immunization and generation of hybridomas followed by screening and validation

• Validation methodology:

  • Use proteolytically-stable Ub-polypeptide conjugates for initial immunization

  • Perform ELISA screens with native iso-peptide linked Ub-polypeptides

  • Validate antibodies in native context using multiple approaches

• Quality control considerations:

  • Acetylate N-terminus of internal sequence peptides to eliminate erroneously recognized positively charged N-terminal amino groups

  • Use screening antigens 2 amino acids longer at N-terminus and/or C-terminus than immunization antigens

  • Include multiple controls to confirm specificity for the ubiquitinated form of UBTF

These approaches are based on strategies developed for other site-specific ubiquitin antibodies like those against H2B-K123ub and huPCNA-K164ub, which could be adapted for UBTF ubiquitination studies .

How can UBTF antibodies be used to analyze transcription factor binding dynamics across different cell types?

For analyzing UBTF binding dynamics across different cell types:

• ChIP-seq comparative analysis:

  • Perform ChIP-seq using UBTF antibodies in multiple cell types

  • Compare genomic binding profiles to identify cell type-specific targets

  • Research has identified differential UBTF binding patterns between mouse NIH3T3 cells and human HMEC cells

• Antibody selection considerations:

  • Choose antibodies validated across the cell types of interest

  • Consider using multiple antibodies to confirm findings

  • The consistency of cell type specificity for UBTF can be assessed when assayed by different antibodies

• Deep learning approaches:

  • Implement methods like SigTFB (Signatures of TF Binding) to identify cell type-specific DNA signatures

  • Analyze hundreds of transcription factors including UBTF to quantify varying degrees of cell type specificity

  • Assess whether UBTF is more cell type specific or general across different cell types

• Motif enrichment analysis:

  • Conduct motif enrichment analysis to understand binding preferences

  • Correlate with gene expression data to establish functional relevance

  • Cell type-specific transcription factors are typically associated with corresponding differences in motif enrichment and gene expression

• Bias correction in ChIP-seq data:

  • Implement weighted analysis of ChIP-seq (WACS) to address biases

  • Use customized weighted controls instead of equally weighted controls for each experiment

  • This approach helps mitigate noise and bias for more accurate binding profiles

These methodologies can help determine whether UBTF binding patterns exhibit cell type specificity or if UBTF functions as a more general transcription factor with conserved binding sites across cell types.

What are the methodological considerations for using UBTF antibodies in cancer biomarker research?

When using UBTF antibodies for cancer biomarker research, particularly in leukemia:

• Patient sample handling:

  • Use standardized protocols for fixation and processing to maintain epitope integrity

  • For AML samples, consider processing bone marrow aspirates within 24 hours

  • Normalize to appropriate controls when comparing UBTF expression levels

• Mutation screening methods:

  • PCR covering exon 13 of UBTF with 6-FAM labeled primers followed by high-resolution fragment analysis to detect UBTF-TDs

  • Sequence PCR products to confirm tandem duplications

  • Targeted NGS-based resequencing to identify co-mutations in UBTF-mutant samples

• Variant allele fraction (VAF) determination:

  • Account for the challenges in establishing accurate VAFs from complex indels

  • Consider tumor purity when interpreting low VAF results

  • Research shows the median VAF of UBTF-TD is approximately 48.0% (range: 9.7%–66.7%)

• PDX model development:

  • Use patient-derived xenograft (PDX) models to study UBTF-TD in vivo

  • Compare UBTF antibody staining patterns between UBTF-TD PDX, KMT2A-r PDX, and normal hematopoietic progenitor cells

  • Research shows UBTF is bound at rDNA in all these cell types, but UBTF was detected at HOXB and HOXA regions only in the UBTF-TD PDX cells

• Therapeutic response monitoring:

  • Use UBTF antibodies to track protein localization and expression during treatment

  • Monitor changes in UBTF binding patterns following menin inhibitor treatment

  • UBTF-TD AMLs show sensitivity to menin inhibitors like SNDX-5613, with treatment resulting in reduced in vitro and in vivo tumor growth