USF2 Antibody, Biotin conjugated

What is USF2 and why is it an important research target?

USF2 (Upstream Stimulatory Factor 2) is a 44 kDa transcription factor belonging to the Myc family characterized by a basic/helix loop helix/leucine zipper domain. It binds to E-box sequences (CACGTG) in promoter regions and regulates the expression of many genes. USF2 has been implicated in several pathological conditions, notably in diabetic nephropathy where it mediates glucose-induced thrombospondin 1 expression and transforming growth factor-β activity in mesangial cells . Its ubiquitous expression and role in transcriptional regulation make it an important target for studying gene expression mechanisms and disease pathways.

How does biotinylation of USF2 antibodies facilitate research applications?

Biotinylation provides several methodological advantages for USF2 antibody applications:

• Enhanced sensitivity: The high-affinity biotin-streptavidin interaction (Kd = 4 × 10^-14 M) allows for signal amplification in detection systems

• Versatility: Permits multiple detection strategies when used with different streptavidin conjugates (HRP, fluorophores)

• Multiplexing capability: Enables dual detection methods when combined with other detection systems

• Stability: Biotin-streptavidin complexes remain stable under various experimental conditions

Biotinylated USF2 antibodies are particularly valuable for immunohistochemistry, Western blotting, ELISA, and chromatin immunoprecipitation experiments studying USF2's role in transcriptional regulation .

What are the critical differences between biotinylation methods for USF2 antibodies?

Different biotinylation methods can significantly impact experimental outcomes with USF2 antibodies:

For detecting nuclear transcription factors like USF2, the ZBPA method is often preferable as it ensures specific labeling that preserves antigen recognition and minimizes background staining in nuclear regions .

How should researchers validate a biotinylated USF2 antibody before experimental use?

A systematic validation approach is necessary:

• Western blot confirmation: Verify that the biotinylated USF2 antibody detects a band at the expected molecular weight (44 kDa) in nuclear extracts or whole cell lysates from appropriate cell lines (e.g., Jurkat, HepG2)

• Comparison with unconjugated antibody: Compare staining patterns between biotinylated and unconjugated versions of the same USF2 antibody to ensure biotinylation has not altered specificity

• Positive and negative controls: Use tissues/cell lines with known USF2 expression levels as controls - HeLa cells and esophageal tissue typically show high USF2 expression

• Peptide competition assay: Perform blocking with the specific immunogenic peptide to confirm specificity for USF2

• Knockout/knockdown validation: Ideally, validate using USF2 knockout or siRNA knockdown samples to confirm antibody specificity

What are the optimal dilution ranges for biotinylated USF2 antibodies in different applications?

Based on experimental data across multiple studies:

These ranges should be optimized for each specific biotinylated USF2 antibody preparation. ZBPA-biotinylated antibodies typically require higher concentrations than Lightning-Link biotinylated antibodies to achieve equivalent signal intensity .

How can I optimize chromatin immunoprecipitation (ChIP) protocols using biotinylated USF2 antibodies?

For successful ChIP experiments targeting USF2 binding to promoter regions:

• Crosslinking optimization: For transcription factors like USF2, use 1% formaldehyde for 10 minutes at room temperature

• Chromatin fragmentation: Sonicate to achieve fragments of 200-500bp, which is optimal for analyzing USF2 binding sites

• Antibody binding:

  • Pre-clear chromatin with streptavidin beads before adding biotinylated USF2 antibody

  • Use 2-5μg of biotinylated USF2 antibody per ChIP reaction

  • Incubate overnight at 4°C with rotation

• PCR primer design: Design primers flanking known USF2 binding sites in promoters of interest (E-box sequences CACGTG)

• Controls:

  • Include input chromatin control (5-10% of starting material)

  • Use IgG-biotin as negative control

  • Include a known USF2 target gene as positive control (e.g., the region covering -713 to -539 of the USF2 promoter itself, which contains an NF-κB site that influences USF2 expression)

How can I resolve high background issues when using biotinylated USF2 antibodies in immunohistochemistry?

Background issues with biotinylated USF2 antibodies often stem from specific methodological problems:

• Non-specific binding from biotinylation method:

  • If using Lightning-Link biotinylated USF2 antibodies, switch to ZBPA biotinylation method to reduce non-specific binding to stabilizing proteins

  • Filter antibody preparations to remove free biotin molecules

• Endogenous biotin interference:

  • Implement biotin blocking steps (use commercial avidin/biotin blocking kit)

  • Pre-block tissues with 0.1% avidin followed by 0.01% biotin

• Nuclear-specific background issues:

  • Include additional blocking with 5% normal serum from the same species as secondary reagent

  • Test longer incubation in blocking solution (2 hours at room temperature)

  • Add 0.1-0.3M NaCl to antibody dilution buffer to reduce ionic interactions

• Streptavidin detection system:

  • Dilute streptavidin-HRP/fluorophore further (1:1000-1:5000)

  • Try streptavidin conjugates from different manufacturers

For USF2 detection specifically, controlling background in nuclear staining is critical since it can mask genuine transcription factor signals .

How should I interpret variations in USF2 detection levels between different tissues and cell types?

Interpretation of USF2 staining patterns should consider:

• Known expression profiles: USF2 shows variable expression across tissues:

  • Ubiquitous expression but higher in actively dividing cells

  • Strong nuclear localization expected in most positive cells

  • Particularly high expression in esophageal tissue and certain cancer cell lines (Jurkat, HepG2)

• Context-dependent regulation: USF2 expression changes in response to:

  • Glycated albumin exposure (increases expression in mesangial cells)

  • High glucose conditions via CREB-dependent mechanisms

  • Disease states (e.g., increased in diabetic nephropathy)

• Technical considerations:

  • Nuclear extraction efficiency varies between tissues and may affect detection

  • Fixation conditions impact nuclear antigen preservation

  • Antibody accessibility to nuclear compartments differs between tissues

When comparing USF2 levels between conditions, always process and stain samples simultaneously and include appropriate positive control tissues/cells with known USF2 expression .

How can biotinylated USF2 antibodies be employed in dual detection systems for protein interaction studies?

Biotinylated USF2 antibodies enable sophisticated protein interaction studies:

• Proximity Ligation Assay (PLA) approach:

  • Use biotinylated USF2 antibody in combination with antibody against potential interaction partner

  • Employ streptavidin-oligonucleotide conjugate for rolling circle amplification

  • This method can detect USF2 interactions with other transcription factors or co-regulators with single-molecule resolution

• Dual immunofluorescence protocol:

  • Combine biotinylated USF2 antibody (detected with streptavidin-fluorophore 1) and another primary antibody (detected with species-specific secondary-fluorophore 2)

  • Critical to use ZBPA-biotinylated USF2 antibody to minimize background when studying nuclear proteins

  • Particularly valuable for examining USF2 co-localization with NF-κB subunits or other transcriptional machinery components

• Sequential ChIP (ChIP-reChIP):

  • First ChIP with biotinylated USF2 antibody

  • Elute complexes under mild conditions

  • Perform second ChIP with antibody against suspected co-factor

  • This approach identifies genomic loci where USF2 and partner proteins simultaneously bind

What are the key experimental considerations when studying USF2 regulation by glycated albumin or other disease-relevant modifications?

When investigating USF2 regulation in disease contexts:

• Stimulation protocol design:

  • For glycated albumin effects on USF2: Use physiologically relevant concentration (200 μg/ml), similar to clinical specimens

  • Control experiments should include non-glycated albumin at equivalent concentrations

  • Time-course experiments (24-72 hours) are essential to capture transcriptional changes

• Measuring transcriptional vs. post-transcriptional regulation:

  • Combine promoter-luciferase assays with mRNA stability assessments (actinomycin D chase)

  • USF2 promoter constructs should include the -837 to -430 region containing the NF-κB binding site for glycated albumin response

• Nuclear translocation and binding dynamics:

  • Assess nuclear accumulation of NF-κB subunits (especially p65) in parallel with USF2 expression changes

  • Employ EMSA and ChIP assays to confirm protein-DNA interactions at specific promoter sequences

  • Mutation of specific binding sites (e.g., NF-κB site) in reporter constructs confirms mechanism

• Validation in appropriate disease models:

  • siRNA knockdown experiments targeting key pathway components (e.g., p65) can confirm mechanistic relationships

  • Translating findings from cell culture to animal models requires careful consideration of tissue-specific effects and appropriate controls

How might advances in site-specific biotinylation technologies improve USF2 antibody performance in single-cell applications?

Emerging technologies for site-specific biotinylation could significantly enhance USF2 antibody applications:

• Enzymatic biotinylation approaches:

  • BirA ligase-mediated site-specific biotinylation of engineered antibody domains

  • Sortase-mediated conjugation at defined C-terminal sequences

  • These approaches yield highly homogeneous biotinylated antibody preparations with preserved antigen binding capacity

• Single-cell applications potential:

  • Enhanced sensitivity for detecting low-abundance USF2 in rare cell populations

  • Improved signal-to-noise ratio for single-cell imaging of transcription factor dynamics

  • Compatibility with multiplexed single-cell profiling methods

• Quantitative considerations:

  • Controlled biotinylation stoichiometry enables more accurate quantification

  • Reduced background facilitates automated image analysis of nuclear USF2 localization patterns

  • Better detection of subtle changes in USF2 levels during cellular responses

ZBPA-based approaches already demonstrate superior specificity compared to traditional methods, but further refinements in bioconjugation chemistry may yield even better performing reagents for studying transcription factors at single-cell resolution .

What methodological adaptations are required when using biotinylated USF2 antibodies in CRISPR-based genomic screening approaches?

Integrating biotinylated USF2 antibodies with CRISPR technologies requires specific methodological considerations:

• CUT&RUN/CUT&Tag adaptations:

  • For mapping USF2 binding sites genome-wide, biotinylated USF2 antibodies can be used with protein A-micrococcal nuclease (pA-MNase) or protein A-Tn5 transposase (pA-Tn5)

  • Critical parameter: Use ZBPA-biotinylated antibodies to ensure specific binding to USF2 and minimize background

  • Optimize digestion/tagmentation conditions for transcription factor footprinting (typically shorter than for histone marks)

• CRISPR screening readouts:

  • For screening gene dependencies affecting USF2 activity:

    • Develop reporter systems based on USF2 binding sites (E-box sequences)

    • Use biotinylated USF2 antibodies to assess protein levels following CRISPR perturbations

    • Combine with RNA-seq to correlate changes in USF2 binding with transcriptional outcomes

• Technical requirements:

  • Permeabilization conditions must be optimized to maintain nuclear architecture while allowing antibody access

  • Fixation protocols need careful balancing to preserve epitopes while stabilizing chromatin

  • Signal amplification strategies may be needed for detecting low-abundance binding events

These approaches can help dissect the complex regulatory networks involving USF2 in various physiological and pathological contexts, particularly in diabetes-related complications where USF2 plays a significant role .