USF2 Antibody, FITC conjugated

What is USF2 and what is its biological function?

USF2 (Upstream Transcription Factor 2, C-Fos Interacting) is a transcription factor that binds to symmetrical DNA sequences called E-boxes (5'-CACGTG-3') found in various viral and cellular promoters . It functions as a regulatory protein in gene expression and is also known as Class B basic helix-loop-helix protein 12 (bHLHb12), FOS-interacting protein (FIP), and Major late transcription factor 2 . USF2 plays critical roles in transcriptional regulation of various cellular processes and has been implicated in several biological pathways including inflammation, cell migration, and proliferation . The full protein consists of 346 amino acids with a calculated molecular weight of 37 kDa, though it is typically observed at approximately 44 kDa in experimental conditions .

What applications is the FITC-conjugated USF2 antibody suitable for?

The FITC-conjugated USF2 antibody is primarily designed for immunofluorescence-based applications. While specific applications may vary based on manufacturer, the antibody has been tested in:

• ELISA (Enzyme-Linked Immunosorbent Assay)

• Immunofluorescence (IF) microscopy

• Potentially useful for flow cytometry due to FITC conjugation

Non-conjugated versions of USF2 antibodies are applicable for:

• Western Blotting (WB)

• Immunohistochemistry (IHC)

• Immunoprecipitation (IP)

For optimal results in each application, titration experiments should be conducted to determine the appropriate dilution for your specific experimental system .

What are the optimal storage conditions for maintaining FITC-conjugated USF2 antibody activity?

The FITC-conjugated USF2 antibody should be stored at -20°C or -80°C upon receipt . To maintain antibody integrity:

• Avoid repeated freeze-thaw cycles that can degrade both antibody function and FITC fluorescence

• For the 20μl size that contains 0.1% BSA, aliquoting is unnecessary for -20°C storage

• The antibody is typically supplied in a buffer containing preservatives (e.g., 0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4)

• When working with the antibody, keep it protected from light to prevent photobleaching of the FITC fluorophore

How should I optimize antibody dilution for immunofluorescence experiments with FITC-conjugated USF2 antibody?

For optimal immunofluorescence results with FITC-conjugated USF2 antibody:

• Begin with a titration experiment using different antibody concentrations (e.g., 1:100, 1:200, 1:500, 1:1000)

• Use consistent cell fixation methods (4% paraformaldehyde is commonly effective)

• Include proper positive controls (cell lines known to express USF2, such as HeLa, Jurkat or HepG2)

• Evaluate signal-to-noise ratio at each dilution

• Consider that the recommended dilution for non-conjugated versions ranges from 1:500-1:2000 for Western blotting, but immunofluorescence often requires higher antibody concentrations

• Document optimal parameters including exposure settings, as FITC can photobleach during imaging

Remember that optimal dilution is sample-dependent and should be determined empirically for each experimental system .

What controls should I include when using USF2 antibody in research experiments?

To ensure reliable and interpretable results with USF2 antibody:

Positive controls:

• Cell lines with confirmed USF2 expression (Jurkat, HepG2, HeLa, 293T)

• Recombinant USF2 protein

Negative controls:

• USF2 knockdown samples (consider using USF2-KD cells as described in recent publications)

• Secondary antibody-only controls (for non-conjugated primary antibodies)

• Isotype controls (rabbit IgG at the same concentration)

• Cells known to have low USF2 expression

Specificity controls:

• Peptide competition assay using the immunogen peptide (amino acids 21-162 of human USF2)

• Western blot validation showing specific band at approximately 44 kDa

How can I assess specificity of the USF2 antibody in my experimental system?

To validate antibody specificity for your particular research application:

• Molecular weight verification: Confirm detection of a single band at approximately 44 kDa by Western blot in appropriate positive control samples

• Knockdown validation: Compare antibody staining between wild-type and USF2 knockdown samples; recent publications demonstrate effective USF2-KD systems that could serve as negative controls

• Immunogen competition: Pre-incubate the antibody with excess immunogen peptide (amino acids 21-162 of human USF2) before staining to block specific binding

• Cross-reactivity assessment: If working with multiple species, verify reactivity with the target species (documented reactivity includes human, mouse, and rat)

• Cellular localization pattern: USF2 should primarily show nuclear localization consistent with its function as a transcription factor

How can I use USF2 antibody to investigate transcription factor binding patterns and genomic distribution?

Recent research using CUT&Tag-seq (Cleavage Under Targets and Tagmentation) has revealed important insights about USF2 genomic distribution . To implement similar approaches:

• ChIP-seq or CUT&Tag protocol optimization:

  • Cross-link protein-DNA complexes using formaldehyde (typically 1%)

  • Sonicate chromatin to appropriate fragment size (200-500bp)

  • Immunoprecipitate with USF2 antibody (may require non-conjugated version)

  • Verify enrichment of known USF2 binding sites by qPCR before sequencing

• Analysis of binding patterns:

  • USF2 binding sites are predominantly enriched in promoter regions (34.84% of binding sites)

  • Focus analysis on E-box motifs (5'-CACGTG-3') which are known USF2 binding sequences

  • Consider co-binding analysis with other transcription factors, as USF2 has been shown to potentially co-regulate genes with other transcription factors in the CAF-C7 gene regulatory network

• Functional validation:

  • Combine binding data with expression analysis following USF2 knockdown

  • Recent studies indicate USF2 may regulate genes involved in retinoic acid metabolism and PI3K-AKT pathway

What is known about USF2's role in epigenetic regulation and histone modifications?

USF2 shows interesting relationships with histone modifications that influence transcription factor binding:

• Histone modification patterns near USF2 binding sites:

  • USF2 belongs to the bHLH family of transcription factors, which display distinctive histone modification (HM) pattern preferences

  • The bHLH family shows conserved HM pattern preferences across different cell lines

  • Analysis techniques like ChIP-seq for both USF2 and histone marks can reveal correlations between binding and specific modifications

• Cell-type specificity:

  • Despite conservation of HM preferences within TF families, binding sites can vary significantly between cell types

  • Studies show that fewer than half of transcription factor binding sites are shared among different cell lines

  • When designing experiments to study USF2-histone modification relationships, consider cell-type specificity

• Experimental approach:

  • Sequential ChIP (ChIP-reChIP) can be used to determine if USF2 binds to regions with specific histone modifications

  • Compare histone modification patterns at USF2 bound versus unbound regions containing E-box motifs

  • Consider the 1kb regions upstream and downstream of binding sites when analyzing histone modification patterns

How does USF2 function relate to cellular phenotypes in cancer and other disease models?

Recent research has uncovered important roles for USF2 in various cellular processes relevant to disease:

• USF2 in cancer-associated fibroblasts (CAFs):

  • USF2 knockdown increases expression of genes involved in inflammatory response, chemotaxis, and cell migration

  • USF2 appears to inhibit fibroblast growth by suppressing TGFβ signaling and activating retinoic acid metabolism

  • USF2 may antagonize RUNX1 function in fibroblast populations, with opposing effects on cell behavior

• Experimental approaches to study USF2 function:

  • Generate USF2 knockdown models using siRNA or CRISPR/Cas9 systems

  • Perform RNA-seq analysis following USF2 manipulation to identify regulated pathways

  • Combine with functional assays for proliferation, migration, and inflammatory response

  • Use immunofluorescence with FITC-conjugated USF2 antibody to track protein localization during cellular phenotype changes

• Disease-specific considerations:

  • USF2 may have context-dependent roles in different disease settings

  • Consider co-staining with markers of specific cellular states or pathways

  • In cancer studies, correlate USF2 expression with patient outcomes or treatment responses

What are common causes of high background when using FITC-conjugated antibodies in immunofluorescence?

High background is a common challenge when working with fluorescently labeled antibodies. To minimize background with FITC-conjugated USF2 antibody:

• Fixation optimization:

  • Test different fixation methods (paraformaldehyde, methanol, or acetone)

  • Overfixation can increase autofluorescence; typically 10-20 minutes with 4% paraformaldehyde is sufficient

• Blocking improvements:

  • Use 3-5% BSA or 5-10% serum from the same species as the secondary antibody (for two-step protocols)

  • Add 0.1-0.3% Triton X-100 for permeabilization

  • Consider adding 0.1% glycine to quench free aldehyde groups after fixation

• Washing optimization:

  • Increase washing steps (at least 3 washes of 5 minutes each)

  • Use PBS with 0.05-0.1% Tween-20 for more efficient washing

• Antibody dilution:

  • Further dilute the antibody if background remains high after optimizing other parameters

  • Filter the diluted antibody solution (0.22μm filter) to remove aggregates

• Autofluorescence reduction:

  • Include a 10-minute treatment with 0.1% Sudan Black B in 70% ethanol after antibody incubation

  • Consider using specialized autofluorescence quenching reagents commercially available

How can I multiplex USF2 detection with other markers in the same sample?

For co-detection of USF2 with other proteins of interest:

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap with FITC (ex/em: 495/519 nm)

  • Good companions include:

    • DAPI for nuclear counterstaining (ex/em: 358/461 nm)

    • Cy3/TRITC for second target (ex/em: 550/570 nm)

    • Cy5/Alexa647 for third target (ex/em: 650/670 nm)

• Antibody compatibility:

  • When using multiple primary antibodies, ensure they are raised in different host species

  • If antibodies are from the same species, use sequential immunostaining with blocking steps between

• Staining protocol:

  • Apply primary antibodies sequentially if there are concerns about cross-reactivity

  • For nuclear transcription factors like USF2, ensure adequate permeabilization (0.2-0.5% Triton X-100)

  • Consider tyramide signal amplification (TSA) for detecting low abundance targets alongside USF2

• Controls for multiplexing:

  • Single-stained controls to establish bleed-through parameters

  • Isotype controls for each primary antibody used

  • Secondary-only controls to evaluate non-specific binding

How do I assess and prevent loss of FITC signal during storage and experimental procedures?

FITC is susceptible to photobleaching and signal decay. To preserve signal integrity:

• Storage considerations:

  • Store the FITC-conjugated antibody in dark containers or wrapped in aluminum foil

  • Maintain recommended storage temperature (-20°C or -80°C)

  • Add antifade reagents if storing stained samples long-term

• During experiments:

  • Minimize exposure to light during all steps

  • Work in reduced ambient lighting when possible

  • Use antifade mounting media containing DABCO or similar compounds

  • Consider newer generation antifade reagents specifically designed for FITC preservation

• Microscopy settings:

  • Use the minimum excitation intensity necessary

  • Employ neutral density filters to reduce excitation light

  • Capture FITC channels first in multi-channel imaging

  • Consider confocal rather than widefield microscopy for reduced photobleaching

• Signal verification:

  • Document initial signal intensity in control samples

  • If signal decay is observed over time, prepare fresh samples or consider alternative conjugates with greater photostability (e.g., Alexa488)

What recent discoveries about USF2 function should inform my experimental design?

Recent research has revealed several important aspects of USF2 function that may influence experimental design:

• Antagonistic relationship with RUNX1:

  • USF2 and RUNX1 appear to have opposing effects on gene expression and cellular function in fibroblasts

  • Consider co-staining or co-analysis of RUNX1 and USF2 in your experimental system

  • USF2 knockdown increases expression of genes involved in inflammatory response and MAPK pathway activation

• Genomic distribution patterns:

  • USF2 binding sites are predominantly found in promoter regions (34.84%)

  • USF2 may coordinate with other transcription factors in the CAF-C7 gene regulatory network

  • E-box motifs (5'-CACGTG-3') are primary USF2 binding sequences

• Pathway involvement:

  • USF2 may regulate genes involved in retinoic acid metabolism and PI3K-AKT pathway

  • USF2 appears to suppress TGFβ signaling in some cellular contexts

  • Consider pathway analysis and functional studies alongside USF2 detection

How can I design experiments to study USF2 involvement in transcription factor networks?

To investigate USF2's role in transcription factor networks:

• Integrative genomic approaches:

  • Combine ChIP-seq or CUT&Tag-seq for USF2 with RNA-seq following USF2 manipulation

  • Identify direct USF2 targets by correlating binding with expression changes

  • Use motif analysis to identify potential co-factors binding near USF2 sites

• Protein-protein interaction studies:

  • Co-immunoprecipitation using USF2 antibodies to identify interacting partners

  • Proximity ligation assay (PLA) to visualize and quantify interactions in situ

  • FRET-based approaches using fluorescently tagged USF2 and candidate interactors

• Gene regulatory network analysis:

  • USF2 has been implicated in the CAF-C7 gene regulatory network

  • Consider computational approaches to reconstruct networks based on multi-omics data

  • Validate key network connections through perturbation experiments