BBC3 Antibody, FITC conjugated

What is BBC3 and why is it an important research target?

BBC3, also known as PUMA (p53 upregulated modulator of apoptosis), is a pro-apoptotic member of the Bcl-2 protein family. The PUMA gene is located at chromosome 19q and encodes a 193-amino acid protein that shares 91% amino acid identity with the murine sequence. BBC3 expression is regulated by the tumor suppressor p53 and plays a critical role in p53-mediated apoptosis. BBC3 encodes two BH3 domain-containing proteins, PUMA-alpha and PUMA-beta, that are produced through alternative first exon usage and are induced following p53 activation . As a key mediator in apoptotic pathways, BBC3/PUMA serves as an important target for cancer research and studies involving cellular death mechanisms.

What does FITC conjugation mean for BBC3 antibodies and how does it function in research applications?

FITC (fluorescein isothiocyanate) is a fluorochrome dye that absorbs ultraviolet or blue light, causing molecules to become excited and emit visible yellow-green light (maximum absorption at 495 nm and emission at 525 nm) . When conjugated to BBC3 antibodies, FITC functions as a reporter tag that allows for direct visualization of BBC3 protein localization within cells or tissues without requiring secondary antibody labeling. The conjugation process involves FITC reacting with free amino groups of the antibody protein to form stable conjugates . This direct labeling approach simplifies experimental workflows, reduces background signal, and enables multicolor immunofluorescence studies when combined with other differently-labeled antibodies.

What experimental applications are FITC-conjugated BBC3 antibodies suitable for?

FITC-conjugated BBC3 antibodies can be utilized in multiple experimental applications:

These applications leverage the direct fluorescence properties of the FITC conjugate, allowing for direct detection of BBC3/PUMA protein in various experimental contexts .

What host species and reactivity profiles are available for FITC-conjugated BBC3 antibodies?

Based on the available research tools, FITC-conjugated BBC3 antibodies typically present the following profiles:

• Host species: Primarily rabbit-derived polyclonal antibodies

• Reactivity: Available options include:

  • Human-specific

  • Human and mouse cross-reactive

  • Human, mouse, and rat cross-reactive

When selecting an antibody for your research, it's critical to match the species reactivity with your experimental model. Cross-reactivity between human and rodent models can be beneficial for translational research projects that span multiple model systems .

How should FITC-conjugated BBC3 antibodies be stored to maintain optimal activity?

Proper storage is essential for maintaining the fluorescence properties and binding capabilities of FITC-conjugated BBC3 antibodies:

• Temperature: Store at 2-8°C for short-term (up to three months) or -20°C for long-term storage

• Critical note: DO NOT FREEZE FITC-conjugated antibodies that are in liquid form, as this can damage the fluorophore structure

• For lyophilized forms: Store at -20°C or -80°C upon receipt, then follow manufacturer guidelines for reconstitution

• After reconstitution: Store at 4°C for up to one month, or aliquot and freeze at -20°C for up to six months

• Avoid repeated freeze-thaw cycles as these significantly reduce antibody activity and fluorescence intensity

• Protect from light exposure, as FITC is photosensitive and can photobleach when exposed to light sources for extended periods

What controls should be included when using FITC-conjugated BBC3 antibodies?

Implementing appropriate controls is essential for validating results obtained with FITC-conjugated BBC3 antibodies:

• Positive control: Use cell lines known to express BBC3/PUMA, such as HepG2, K562, or HeLa cells, which have been validated for BBC3 expression

• Negative control: Include samples from BBC3 knockout tissues/cells or use isotype control antibodies (FITC-conjugated rabbit IgG from non-immunized animals)

• Autofluorescence control: Unstained samples to account for natural cellular fluorescence in the FITC channel

• Blocking control: Pre-incubate the antibody with its specific immunogen peptide to confirm binding specificity

• Cross-reactivity control: When working with human-derived samples, include mouse or rat samples (depending on the antibody's specified cross-reactivity) to confirm species specificity

What is the molecular weight of BBC3/PUMA protein that should be detected using these antibodies?

When performing Western blot analysis using BBC3 antibodies, researchers should be aware of the different isoforms and their corresponding molecular weights:

• The observed molecular weight of the primary BBC3/PUMA band is approximately 18-26 kDa

• The calculated molecular weight based on the amino acid sequence is approximately 26.5 kDa

• Some antibodies may detect multiple bands representing different BBC3 isoforms:

  • PUMA-alpha and PUMA-beta are produced through alternative first exon usage

  • Additional bands at higher molecular weights may represent post-translationally modified forms of the protein

  • Validation experiments confirm bands at approximately 18 kDa in human HepG2, K562, and HeLa cell lysates

How can one optimize FITC-conjugated BBC3 antibody performance in multicolor flow cytometry experiments?

Optimizing FITC-conjugated BBC3 antibodies for multicolor flow cytometry requires careful consideration of several technical factors:

• Compensation strategy:

  • FITC has spectral overlap with PE and other fluorophores in the green-yellow spectrum

  • Perform single-color controls with FITC-conjugated BBC3 antibody to establish proper compensation matrices

  • Consider using brightest level of FMO (Fluorescence Minus One) controls for accurate gating

• Antibody titration optimization:

  • Test dilutions ranging from 1:50 to 1:1000 to determine the optimal signal-to-noise ratio

  • Plot staining index (mean positive - mean negative/2× SD of negative) against antibody concentration to identify optimal usage

• Buffer composition considerations:

  • Use buffers containing 0.1-0.5% BSA to reduce non-specific binding

  • Include 0.01-0.1% sodium azide to preserve antibody stability during staining

  • For intracellular BBC3 detection, use permeabilization buffers containing 0.1% saponin or 0.3% Triton X-100

• Sample preparation protocol:

  • For intracellular BBC3 detection, ensure complete fixation (4% paraformaldehyde for 15 minutes)

  • Allow sufficient permeabilization time (typically 10-15 minutes) for antibody access to intracellular BBC3

  • When studying apoptosis, collect both attached and floating cells to capture the complete apoptotic population

• Excitation/emission parameters:

  • Use 488 nm laser for optimal FITC excitation

  • Collect emission through a 530/30 nm bandpass filter

  • Adjust PMT voltage to place negative population in first decade of log scale

What are the key considerations when using FITC-conjugated BBC3 antibodies in fixed versus live-cell imaging applications?

The application of FITC-conjugated BBC3 antibodies differs significantly between fixed and live-cell imaging scenarios:

Fixed Cell Applications:

• Fixation method impact: Paraformaldehyde (4%) preserves FITC fluorescence better than methanol-based fixatives

• Epitope accessibility: The BH3 domain epitope of BBC3 may require enhanced permeabilization for optimal antibody penetration

• Mounting media selection: Use anti-fade mounting media with DAPI to reduce photobleaching and provide nuclear counterstaining

• Signal amplification: Not typically needed due to direct FITC conjugation, but tyramide signal amplification can be employed for low-abundance targets

Live Cell Applications:

• Cell permeability challenges: FITC-conjugated antibodies cannot penetrate live cell membranes without delivery systems

• Potential applications: Limited to extracellular epitopes or requires specialized delivery methods (microinjection, cell-penetrating peptides)

• Phototoxicity considerations: Minimize exposure times as FITC excitation can generate reactive oxygen species damaging to live cells

• Alternative approaches: Consider using FITC-conjugated Fab fragments which are smaller and may cause less interference with protein function

For most BBC3/PUMA research applications, fixed-cell approaches are recommended as BBC3 is primarily an intracellular protein involved in mitochondrial-associated apoptotic pathways .

How do different FITC-to-protein ratios affect the performance of BBC3 antibodies in various applications?

The FITC-to-protein (F/P) ratio is a critical parameter that significantly impacts antibody performance:

Optimally labeled antibodies typically have F/P ratios between 3-5 FITC molecules per antibody molecule. Fluorescent labeling of antibodies with high F/P ratios (>6) typically results in increased non-specific binding and decreased quantum yield due to the self-quenching effect . For BBC3 detection, medium F/P ratios (3-5) generally provide the best balance of sensitivity and specificity.

What are the most effective fixation and permeabilization protocols for detecting BBC3 with FITC-conjugated antibodies in different cell types?

Optimization of fixation and permeabilization protocols is essential for successful BBC3 detection across different cell types:

For Adherent Cell Lines (e.g., HeLa, HepG2):

• Fixation: 4% paraformaldehyde in PBS for 15 minutes at room temperature

• Permeabilization: 0.1% Triton X-100 in PBS for 10 minutes

• Blocking: 5% normal serum (matching secondary antibody host) with 0.1% Tween-20 in PBS for 1 hour

• Antibody incubation: FITC-conjugated BBC3 antibody (1:50-1:100) in blocking buffer overnight at 4°C

For Suspension Cells (e.g., K562, Lymphocytes):

• Fixation: 2% paraformaldehyde in PBS for 10 minutes at room temperature

• Permeabilization: 0.1% saponin in PBS with 0.5% BSA for 15 minutes

• Blocking: 2% BSA with 0.1% saponin in PBS for 30 minutes

• Antibody incubation: FITC-conjugated BBC3 antibody (1:100) in blocking buffer for 2 hours at room temperature

For Tissue Sections:

• Fixation: Already fixed in formalin and paraffin-embedded, or fresh-frozen sections fixed in ice-cold acetone

• Antigen retrieval: Citrate buffer (pH 6.0) heat-induced epitope retrieval for 20 minutes (for FFPE tissues)

• Permeabilization: 0.2% Triton X-100 in PBS for 15 minutes

• Blocking: 10% normal serum with 1% BSA in PBS for 1-2 hours

• Antibody incubation: FITC-conjugated BBC3 antibody (1:50) in blocking buffer overnight at 4°C

These protocols may require further optimization based on specific cell types and research questions .

How can researchers troubleshoot weak or absent signals when using FITC-conjugated BBC3 antibodies?

When encountering weak or absent signals with FITC-conjugated BBC3 antibodies, consider the following troubleshooting approaches:

• Protein expression verification:

  • Confirm BBC3/PUMA expression in your experimental system using RT-PCR or Western blot

  • BBC3 expression is typically induced by p53 activation; consider using positive control treatments like DNA-damaging agents

• Technical optimization:

  • Antibody concentration: Test higher concentrations (1:25 instead of 1:100)

  • Incubation conditions: Extend incubation time to overnight at 4°C

  • Permeabilization enhancement: Increase Triton X-100 concentration to 0.3% or extend permeabilization time

• Signal amplification strategies:

  • Anti-FITC antibody: Use an anti-FITC antibody conjugated to a brighter fluorophore

  • Biotin-streptavidin system: If a biotinylated anti-FITC is available, use with fluorescent streptavidin

  • TSA (Tyramide Signal Amplification): Compatible with FITC detection for significant signal enhancement

• Fluorescence preservation:

  • Minimize light exposure during all procedures

  • Use fresh anti-fade mounting media

  • Image samples promptly after preparation

• Epitope accessibility issues:

  • The BH3 domain epitope of BBC3 may be masked by protein interactions

  • Try alternative fixation methods that may better preserve epitope structure

  • Consider denaturing conditions that might expose the epitope more effectively

What is the relationship between BBC3 antibody epitope selection and functional studies of apoptosis?

The epitope targeted by BBC3/PUMA antibodies significantly impacts functional studies of apoptosis:

BH3 Domain Epitope Antibodies:

• Target the functionally critical BH3 domain of BBC3/PUMA

• Ideal for mechanistic studies focusing on protein-protein interactions

• May be blocked when BBC3 is bound to anti-apoptotic Bcl-2 family members

• Particularly useful for studying available/unbound BBC3 protein

N-terminal Epitope Antibodies:

• Target sequences at the N-terminus of human PUMA/BBC3 (e.g., amino acids 5-23)

• Less likely to be obscured by protein interactions

• Better for quantitative studies of total BBC3 expression

• May recognize both PUMA-alpha and PUMA-beta isoforms

For apoptosis pathway studies, researchers should consider using complementary antibodies targeting different epitopes to distinguish between bound and unbound BBC3, or to differentiate between specific isoforms. When designing experiments to study p53-dependent apoptosis mediated by BBC3, antibodies targeting the BH3 domain provide direct insight into the functional portion of the protein responsible for interacting with anti-apoptotic Bcl-2 family members .

How can FITC-conjugated BBC3 antibodies be incorporated into multiplexed imaging approaches for studying apoptotic pathways?

Integrating FITC-conjugated BBC3 antibodies into multiplexed imaging strategies requires careful fluorophore selection and protocol optimization:

• Compatible fluorophore combinations:

  • FITC (BBC3) + Cy3/TRITC (Bcl-2) + Cy5 (active caspase-3) + DAPI (nuclei)

  • This combination allows visualization of the complete apoptotic cascade from BBC3 activation to downstream effector activation

• Sequential staining protocol for four-color imaging:

  • Begin with FITC-conjugated BBC3 antibody staining

  • Block with excess unconjugated rabbit IgG

  • Continue with other primary antibodies from different host species

  • Complete with species-specific secondary antibodies for non-FITC primaries

• Image acquisition strategy:

  • Capture FITC channel first (most susceptible to photobleaching)

  • Use narrow bandpass filters to minimize spectral overlap

  • Apply linear unmixing algorithms if spectral overlap occurs

  • Consider confocal microscopy for improved resolution of subcellular localization

• Advanced multiplexing approach:

  • Combine with phospho-p53 staining to correlate p53 activation with BBC3 upregulation

  • Add mitochondrial markers (e.g., TOMM20) to assess BBC3 translocation to mitochondria

  • Include cytochrome c staining to visualize mitochondrial outer membrane permeabilization

• Quantitative analysis workflow:

  • Measure BBC3 intensity relative to mitochondrial markers

  • Correlate BBC3 levels with downstream apoptotic markers

  • Create ratiometric measurements of pro- versus anti-apoptotic proteins

This multiplexed approach enables comprehensive visualization of the p53-BBC3-mitochondria-caspase axis in apoptotic research .

What are the advantages and limitations of using FITC-conjugated BBC3 antibodies in pH-dependent experimental systems?

FITC fluorescence characteristics are notably pH-sensitive, creating both opportunities and challenges for BBC3 research:

Advantages in pH-Dependent Systems:

• pH indicators: FITC fluorescence intensity increases at higher pH (6.5-9.0), making it useful for simultaneous BBC3 detection and cellular pH monitoring

• Tumor microenvironment studies: Can be utilized in cancer research to correlate BBC3 expression with changes in tumor acidity

• Selective visualization: When combined with pH-sensitive targeting approaches like pHLIP conjugates, can enable selective visualization in acidic tumor environments

• Mitochondrial membrane potential studies: Changes in mitochondrial pH during apoptosis can be monitored alongside BBC3 translocation

Limitations and Considerations:

• pH sensitivity: FITC fluorescence is significantly reduced at acidic pH (<6.0), potentially causing false-negative results in acidic cellular compartments

• Experimental design constraints: Buffer systems must be carefully controlled, ideally maintaining pH 7.2-7.4 for consistent results

• Mitochondrial acidification: During apoptosis, mitochondria can become more acidic, potentially reducing FITC signal intensity at this critical location

• Alternative approaches: Consider pH-stable fluorophores (Alexa Fluor 488) for experiments involving significant pH fluctuations

• Quantitative analysis challenges: Fluorescence intensity variations due to pH must be distinguished from actual changes in BBC3 protein levels

For experiments specifically studying BBC3 in varied pH environments (such as tumor microenvironments or organelles with distinct pH), careful calibration controls must be included, or alternative pH-stable fluorophores should be considered .