BG4 Antibody

What is BG4 antibody and what DNA structures does it recognize?

BG4 is a monoclonal single-chain variable fragment (scFv) antibody generated by phage display that specifically targets G-quadruplex structures. It was developed to recognize a broad range of G4 configurations with high affinity and specificity. BG4 preferentially binds to G-rich DNA that forms G-quadruplexes, particularly when they adopt parallel orientation, while showing minimal interaction with complementary C-rich or random sequences .

BG4 has demonstrated remarkable selectivity for G-quadruplex structures over other DNA conformations. Importantly, the mere presence of a G4-motif within duplex DNA is insufficient for antibody recognition - the G-quadruplex structure must be properly formed . This structural specificity makes BG4 an invaluable tool for studying G4 dynamics in various experimental contexts.

What are the primary research applications of BG4?

BG4 has been successfully employed across multiple experimental platforms:

• Immunofluorescence (IF): For visualizing G4 structures within cellular nuclei and investigating their distribution throughout the cell cycle

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative binding studies and affinity measurements

• Chromatin Immunoprecipitation (ChIP-seq): For genome-wide identification of G4 structures within chromatin

• Single-molecule analysis: Including single-molecule pull-down (SiMPull) for examining G4 conformational dynamics

• Atomic Force Microscopy (AFM): For structural characterization of BG4-G4 complexes

These diverse applications have enabled researchers to detect over 10,000 G4 structures in regulatory, nucleosome-depleted regions of human chromatin, significantly advancing our understanding of G4 biology .

How does BG4 compare to other G-quadruplex binding antibodies?

Several G4 structure-specific antibodies have been developed, including HF1, hf2, and 1H6, alongside BG4. Among these, BG4 has gained widespread adoption due to its exceptional properties:

• Affinity: BG4 exhibits impressive binding affinity with a Kd of approximately 2.0 nM as reported in early characterization studies, which is among the highest for G4-binding antibodies .

• Specificity: BG4 demonstrates superior specificity for G4 structures, particularly those in parallel orientation .

• Versatility: Unlike some G4 antibodies that recognize more limited subsets of G4 conformations, BG4 can bind to a broader range of G4 topologies .

• Validation: BG4 has been extensively characterized using multiple biochemical and biophysical techniques, providing researchers with high confidence in its specificity .

This combination of properties has made BG4 the preferred antibody for many G-quadruplex research applications.

How does BG4 binding affinity vary with different G-quadruplex structures?

BG4 exhibits differential binding affinities depending on G-quadruplex structural characteristics:

The ability of BG4 to bind to a range of G4 structures with different affinities makes it a versatile tool for studying diverse G4 conformations in various genomic contexts.

Can BG4 recognize partially folded or modified G-quadruplex structures?

One of the most significant research findings is that BG4 can indeed recognize partially folded G-quadruplex structures and those containing destabilizing modifications:

• Single guanine substitutions: BG4 maintains binding capability, albeit with reduced affinity, when a single guanine is substituted with 8-aza-7-deaza-G, T, A, or C. The degree of affinity reduction varies depending on the location and base type .

• Damaged bases: BG4 can recognize G-quadruplexes containing damaged bases such as 8-oxoguanine and O6-methylguanine, although with reduced affinity depending on lesion location .

• Multiple substitutions: When two guanine substitutions are present in a telomeric construct, BG4 binding is dramatically reduced or abolished completely .

This unexpected flexibility in substrate recognition has important implications for detecting G4 structures in genomic regions with mutations or DNA damage.

What is the stoichiometry of BG4 binding to G-quadruplexes?

Atomic force microscopy (AFM) studies have revealed that BG4 binds to telomeric G-quadruplex substrates with a 1:1 stoichiometry . This binding ratio is consistent across different telomeric G-quadruplex constructs, suggesting a single binding site on the G-quadruplex structure for BG4 interaction.

Understanding this stoichiometry is crucial for accurately interpreting quantitative data from BG4-based assays and for designing appropriate controls and calibration standards in experimental workflows.

Does BG4 binding influence G-quadruplex stability?

An intriguing property of BG4 is its ability to affect G-quadruplex dynamics:

• Promotion of folding: Single-molecule FRET assays have demonstrated that BG4 binding promotes folding of telomeric G-quadruplexes harboring a single G to T substitution or 8-oxoguanine .

• Stabilization effect: BG4 binding appears to enhance the stability of partially folded G-quadruplex structures, potentially shifting the equilibrium toward the folded state .

This stabilizing effect must be considered when interpreting BG4-based cellular imaging data, as the observed G4 patterns may partly reflect BG4-induced stabilization rather than the native G4 landscape alone.

What is the recommended protocol for BG4 validation and immunofluorescence?

Before using BG4 for cellular imaging, validation of antibody activity is essential:

• Purity assessment: SDS-PAGE with Coomassie staining to ensure high purity of BG4 preparation .

• Activity validation: Treatment of cells (e.g., HeLa LT cells) with G-quadruplex-stabilizing ligands such as pyridostatin (PDS, 10 μM overnight) should significantly increase the number of nuclear BG4 foci compared to untreated controls .

• Immunofluorescence procedure:

  • Fix cells with appropriate fixative (typically paraformaldehyde)

  • Permeabilize cell membranes

  • Block with suitable blocking buffer

  • Incubate with purified BG4 (concentration optimized for your specific application)

  • Detect bound BG4 using anti-FLAG antibodies (if using FLAG-tagged BG4)

  • Counterstain nuclei and visualize using fluorescence microscopy

Recommended controls include samples lacking either BG4 or the secondary detection antibody, as well as competitive binding with known G4 ligands to validate signal specificity.

How should BG4 be prepared and purified for optimal results?

For optimal performance, BG4 preparation should include:

• Expression system: BG4 can be expressed from the available plasmid (Addgene #55756) .

• Purification steps:

  • Standard protein purification techniques applicable to scFv antibodies

  • Addition of size-exclusion chromatography step to enhance BG4 purity and remove residual nucleic acids that might interfere with substrate binding

  • Quality control via SDS-PAGE to confirm purity

The Balasubramanian lab provides detailed protocols for expression and purification, available from their website . It's important to note that contaminating nucleic acids can interfere with substrate binding, single-molecule analysis, and AFM imaging, making thorough purification essential.

What is the recommended protocol for ELISA using BG4?

For quantitative assessment of BG4 binding to G-quadruplex structures, the following ELISA protocol is recommended:

• Sample preparation:

  • Resuspend oligonucleotides at 100 μM in DEPC-filtered water

  • Anneal and fold oligonucleotide substrates properly before use

• ELISA procedure:

  • Use NeutravidinTM-coated 96-well clear polystyrene plates

  • Wash plates three times with ELISA wash buffer

  • Dilute folded oligonucleotide substrates to 10 nM in ELISA assay buffer

  • Add 100 μl of substrate to wells and incubate for 1 hour at room temperature with gentle rocking

  • Wash three times

  • Block wells for one hour with 150 μl blocking buffer (1% BSA, 0.05% Triton X-100, in PBS)

  • Prepare BG4 dilutions (0.1 to 40 nM) in ELISA assay buffer

  • Add 100 μl BG4 dilutions to wells and incubate for one hour

  • Wash three times

  • Add HRP-conjugated anti-FLAG tag antibody (1:50,000 dilution) for 30 minutes

  • Wash three times

  • Develop with 100 μl TMB chromogen for 4 minutes

  • Stop reaction with 100 μl ELISA stop solution

  • Measure absorbance at 450 nm

This protocol allows for quantitative determination of binding affinities and comparison between different G-quadruplex structures.

How can I use BG4 in single-molecule pull-down (SiMPull) assays?

For researchers interested in single-molecule analysis of G-quadruplex structures:

• Surface preparation:

  • Immobilize anti-FLAG antibodies on imaging surface

• BG4 immobilization:

  • Add diluted (1-5 nM) FLAG-tagged BG4 antibody to the antibody-coated surface

  • Incubate for 10 minutes at room temperature

  • Wash sample chamber

• DNA substrate addition:

  • Apply 10 nM non-biotinylated fluorescently labeled (Cy3 or Cy3/Cy5) DNA

  • Incubate for 15 minutes

  • Wash out unbound DNA

• Imaging:

  • Add imaging buffer containing oxygen scavenging system

  • Use appropriate laser excitation (e.g., 532 nm for Cy3)

  • Record multiple short movies from different imaging surfaces

• Data analysis:

  • Analyze with MATLAB script to calculate binding ratio

  • Include controls lacking either anti-FLAG antibody or BG4 protein

  • Include standard samples that form stable G-quadruplexes

This approach enables direct visualization of BG4-G4 interactions at the single-molecule level, providing insights into binding dynamics and conformational changes that bulk assays cannot resolve.

How do results from different BG4 binding assays compare?

There is generally good correlation between bulk biochemical assays and single-molecule techniques for assessing BG4 binding to G-quadruplex structures:

The discrepancy observed with poly-thymidine controls may be explained by differences in experimental setup: in ELISA, biotinylated constructs are bound to plates with restricted movement, whereas in SiMPull, BG4 protein is attached to the slide via a FLAG antibody .

For most G-quadruplex structures, both assays yield comparable results, suggesting either method is suitable for assessing BG4 interactions, with the choice depending on the specific research questions and available equipment.

What factors can affect BG4 binding in cellular assays?

Several factors can influence BG4 binding in cellular contexts:

• Cell cycle phase: Maximum BG4 foci are observed during S phase, suggesting replication-dependent formation of G-quadruplexes .

• G-quadruplex stabilizing ligands: Treatment with compounds like pyridostatin (PDS) increases the number of BG4 foci in cells .

• G-quadruplex resolving enzymes: Knockdown of G4-resolvase proteins such as WRN can modulate the number of BG4 foci within cells .

• Fixation methods: Different cell fixation protocols may affect the preservation and accessibility of G-quadruplex structures.

• BG4 concentration: Titration experiments may be necessary to determine optimal antibody concentration for specific cell types.

Understanding these factors is crucial for accurate interpretation of cellular BG4 staining patterns and for designing appropriate experimental controls.

What are important considerations when comparing BG4 with newer G-quadruplex detection tools?

When evaluating BG4 against other G-quadruplex detection tools:

• Antibody specificity: BG4 specifically recognizes parallel G-quadruplex orientation, whereas other antibodies or tools may detect different conformations .

• Dynamic effects: Unlike some chemical probes, BG4 can promote folding of partially formed G-quadruplexes, potentially altering the native G4 landscape .

• Live cell detection: BG4 requires cell fixation, whereas newer tools like SiR-PyPDS allow real-time detection of G4 in live cells .

• Alternative formats: Researchers should be cautious about IgG derivatives of BG4 available from some suppliers, which use alternative tags and may not have the same performance as the original BG4 .

• Newer alternatives: Consider newer tools like the SG4 nanobody, a camelid heavy-chain-only derived nanobody with low nanomolar affinity for folded G4 structures .

Each detection method has strengths and limitations, and combining multiple approaches often provides the most comprehensive understanding of G-quadruplex biology.

What are emerging applications of BG4 in disease-related research?

BG4 antibody is increasingly being applied to understand the role of G-quadruplexes in various disease processes:

• Cancer research: Investigating G-quadruplex formation in oncogene promoters and their potential as therapeutic targets

• Neurological disorders: Exploring G-quadruplex roles in repeat expansion disorders

• DNA damage response: Studying how damaged bases within G-quadruplexes affect genome stability and repair processes

• Telomere biology: Examining G-quadruplex dynamics at telomeres during cellular aging and in cancer cells

As BG4 methodology becomes more standardized, its application in translational and clinical research contexts is expected to expand significantly.

What methodological improvements might enhance BG4-based G-quadruplex detection?

Several technological advancements could improve BG4-based G-quadruplex research:

• Engineered BG4 variants: Development of BG4 derivatives with altered specificity profiles or enhanced affinity for specific G-quadruplex conformations

• Multimodal imaging: Combining BG4 immunofluorescence with other cellular markers to understand G-quadruplex biology in specific cellular compartments or processes

• Super-resolution microscopy: Application of techniques such as STORM or PALM to achieve nanoscale resolution of BG4-labeled G-quadruplex structures

• Quantitative analysis pipelines: Development of standardized image analysis workflows for quantifying BG4 foci across experimental conditions

• In situ structural analysis: Combining BG4 detection with methods that reveal G-quadruplex conformational states within cellular contexts

These methodological improvements would address current limitations and expand the utility of BG4 in G-quadruplex research.