BCL7C Antibody

What is BCL7C and what is its biological function?

BCL7C is a 23 kDa protein (217 amino acids) that belongs to the BCL7 family. This gene was identified based on its similarity to the N-terminal region of BCL7A protein, which is known to be involved in a three-way gene translocation in a Burkitt lymphoma cell line . While initially thought to play an anti-apoptotic role , recent research has demonstrated that BCL7C functions as a tumor suppressor in ovarian cancer by counteracting mutant p53 activity .

BCL7C is part of the BCL7 family that was first discovered when BCL7A was found to be involved in complex translocations in lymphoma. Each family member appears to have distinct roles in cancer biology, with BCL7C specifically showing tumor suppressor functionality through its interaction with mutant p53 proteins .

What applications are BCL7C antibodies validated for?

BCL7C antibodies are validated for multiple research applications, with specific validations varying by product:

• Western Blot (WB): The most commonly validated application across products

• ELISA: Many antibodies show reactivity in enzyme-linked immunosorbent assays

• Immunofluorescence (IF): Select antibodies are validated for cellular localization studies

• Immunohistochemistry (IHC): Both paraffin-embedded (IHC-P) and frozen section (IHC-F) applications

The specific application validation depends on the antibody source and clone. For instance, the rabbit polyclonal antibody ab231278 is validated for Western blot at 1/1000 dilution using human samples , while mouse monoclonal antibodies like clone 1A4 are validated for Western blot, ELISA, and immunofluorescence applications .

What are the common reactivity profiles of available BCL7C antibodies?

BCL7C antibodies show varying reactivity profiles depending on their source and production method:

When selecting an antibody, it's essential to verify the claimed reactivity against your species of interest. For cross-species applications, antibodies targeting conserved regions of BCL7C typically show broader reactivity .

What are optimal protocols for Western blot analysis of BCL7C?

To achieve optimal Western blot results with BCL7C antibodies, follow these methodological guidelines:

Sample preparation:

• Use nuclear extracts from appropriate cell lines (e.g., HeLa cells are recommended)

• Load approximately 40 μg of protein extract per lane

• Ensure complete denaturation of samples

Protocol parameters:

• Dilution: Most BCL7C antibodies work optimally at 1/1000 dilution for Western blot

• Dilution buffer: Use TBS-Tween containing 5% skimmed milk

• Expected band size: 23 kDa (predicted molecular weight of BCL7C)

• Secondary antibody: Select based on host species (anti-rabbit or anti-mouse)

Controls:

• Positive control: HeLa nuclear extract is commonly used

• Negative control: Consider BCL7C-knockdown samples or non-expressing tissues

• Blocking peptide: If available, use to confirm band specificity

When troubleshooting, remember that BCL7C is a relatively small protein (23 kDa), so standard gel percentage (10-12%) should be appropriate for good resolution .

How can I validate the specificity of BCL7C antibodies?

Proper validation of BCL7C antibodies is critical for reliable research outcomes. Implement these methodological approaches:

• Genetic validation approaches:

  • siRNA knockdown: Transfect cells with BCL7C-specific siRNAs and confirm reduced antibody signal in Western blot or immunofluorescence

  • Overexpression: Transfect cells with tagged BCL7C constructs and verify signal increase

  • CRISPR-Cas9 knockout: Create BCL7C knockout cell lines as definitive negative controls

• Biochemical validation:

  • Blocking peptide experiments: Pre-incubate antibody with immunizing peptide to abolish specific signal

  • Multiple antibody comparison: Test different antibodies targeting distinct BCL7C epitopes

  • Molecular weight verification: Confirm detection at the expected 23 kDa size

• Control samples:

  • Tissue panel analysis: Test expression across tissues with known differential BCL7C expression

  • Species cross-reactivity: Verify specificity across claimed species reactivity

  • Family member specificity: Test for cross-reactivity with related BCL7A and BCL7B proteins

These validation steps should be documented and included when publishing research using BCL7C antibodies to ensure data reproducibility and reliability.

What cell lines are recommended for studying BCL7C expression and function?

Based on the research literature, the following cell lines are recommended for BCL7C studies:

For basic expression analysis:

• HeLa cells: Human cervical adenocarcinoma cell line used for antibody validation

For functional studies (especially p53-related mechanisms):

• TOV112D: Ovarian cancer cells containing mutant p53-R175H

• ES-2: Ovarian cancer cells with mutant p53-S241F

• SKOV3: p53-negative ovarian cancer cells (useful as negative controls)

• A2780: Wild-type p53 ovarian cancer cells

• OVCA420: Ovarian cancer cells used in invasion assays with BCL7C

Experimental approach considerations:

• For studying BCL7C's tumor suppressor function, use cells with mutant p53 (e.g., TOV112D, ES-2)

• For negative controls, use p53-null cells like SKOV3

• For pathway-specific studies, consider using isogenic cell lines differing only in p53 status

These cell lines provide valuable models for investigating BCL7C's biological functions, particularly its interaction with mutant p53 and role in cancer progression.

How does BCL7C interact with mutant p53 proteins and what is the functional significance?

Recent research has revealed a critical interaction between BCL7C and mutant p53 proteins with significant functional implications:

Interaction mechanism:

• Co-immunoprecipitation experiments demonstrate that BCL7C physically interacts with multiple mutant p53 (mtp53) variants, including mtp53-R175H, mtp53-Y220C, mtp53-R248W, mtp53-R249S, and mtp53-R273H

• This interaction forms the molecular basis for BCL7C's tumor suppressor function

Functional consequences:

• Suppression of mutant p53 target genes: BCL7C represses the expression of multiple mutant p53 target genes specifically in cells containing mutant p53, but not in p53-negative cells

• Inhibition of cancer cell proliferation:

  • Ectopic BCL7C markedly inhibits proliferation of cancer cells with mutant p53

  • Knockdown of BCL7C increases proliferation of these cells

  • This effect is p53-dependent as demonstrated in isogenic cell lines

• Suppression of cancer cell invasion:

  • BCL7C expression inhibits invasion of ES-2 and OVCA420 ovarian cancer cells

  • This effect is abrogated when mutant p53 is depleted by siRNA

  • BCL7C has no effect on invasion in cells lacking mutant p53

These findings establish BCL7C as a novel tumor suppressor that acts by directly counteracting the oncogenic activities of mutant p53. This represents a significant advance in understanding how BCL7C contributes to cancer biology and suggests potential therapeutic strategies targeting this interaction in cancers harboring mutant p53 .

What methodologies can be used to study BCL7C's role in tumor suppression?

To investigate BCL7C's tumor suppressive functions, researchers should consider these methodological approaches:

Expression analysis in clinical samples:

• Immunohistochemistry to analyze BCL7C expression in tumor microarrays

• Correlation of expression with clinical parameters (survival, stage, treatment response)

• Prognostic analysis based on BCL7C levels (high BCL7C is associated with favorable prognosis in ovarian cancer)

Molecular interaction studies:

• Co-immunoprecipitation to confirm BCL7C interaction with mutant p53 variants

• ChIP assays to investigate whether BCL7C affects mutant p53 binding to target gene promoters

• Proximity ligation assays to visualize BCL7C-p53 interactions in situ

Functional assays:

• Cell proliferation assays (e.g., CCK-8) following BCL7C overexpression or knockdown

• Cell invasion assays to assess metastatic potential modulation

• Colony formation assays to evaluate long-term growth effects

• Gene expression analysis of mutant p53 target genes after BCL7C manipulation

In vivo studies:

• Xenograft models comparing tumor growth with variable BCL7C expression

• Metastasis models to assess BCL7C's impact on cancer dissemination

• Patient-derived xenografts to evaluate clinical relevance

Research findings indicate that BCL7C suppresses ovarian cancer growth and invasion specifically by inactivating mutant p53. When designing experiments, it's crucial to consider the p53 status of your model system, as BCL7C's tumor suppressive effects are dependent on the presence of mutant p53 .

What are the advantages and limitations of different types of BCL7C antibodies?

Different types of BCL7C antibodies offer distinct advantages and limitations that researchers should consider when selecting reagents:

Polyclonal BCL7C antibodies:

Advantages:

• Recognition of multiple epitopes, providing robust detection even if some epitopes are modified or masked

• Often produce stronger signals due to multiple antibody binding

• Useful for detecting low-abundance proteins or in applications where signal amplification is needed

• Generally more tolerant to minor protein denaturation or fixation-induced changes

Limitations:

• Batch-to-batch variability requiring validation between lots

• Potential for higher background due to non-specific binding

• May cross-react with related proteins (BCL7A, BCL7B)

• Less suitable for applications requiring precise epitope targeting

Monoclonal BCL7C antibodies:

Advantages:

• Consistent epitope recognition with minimal batch variation

• Typically produce cleaner results with lower background

• More suitable for quantitative applications due to consistent binding

• Better for applications like flow cytometry or immunoprecipitation

• Specific clones like 1A4 and 5F1 are validated for multiple applications

Limitations:

• Single epitope recognition may result in false negatives if that epitope is masked

• May be more sensitive to fixation conditions that alter epitope structure

• Generally more expensive than polyclonal antibodies

• May have more limited application validation

Application-specific considerations:

• For Western blot: Both types work well, with polyclonals often giving stronger signals

• For immunoprecipitation: Monoclonals may provide cleaner pull-downs with less background

• For immunohistochemistry: Consider epitope sensitivity to fixation when selecting antibody

• For co-localization studies: Monoclonals may provide more precise subcellular localization

Why might I be getting non-specific bands in Western blot with BCL7C antibodies?

Non-specific bands in Western blots with BCL7C antibodies can arise from several sources. Here are methodological solutions:

Common causes and solutions:

• Antibody specificity issues:

  • Problem: Cross-reactivity with BCL7A or BCL7B family members

  • Solution: Use antibodies validated for specific BCL7C detection; consider monoclonal antibodies targeting unique BCL7C epitopes

• Sample preparation problems:

  • Problem: Protein degradation creating fragments that generate multiple bands

  • Solution: Add protease inhibitors; prepare fresh samples; ensure proper denaturation

• Blocking and washing issues:

  • Problem: Insufficient blocking leading to non-specific binding

  • Solution: Optimize blocking with 5% skimmed milk in TBS-Tween as recommended ; increase washing frequency and duration

• Antibody concentration:

  • Problem: Too concentrated primary antibody causing background binding

  • Solution: Titrate antibody concentration; start with manufacturer's recommended dilution (typically 1:1000) and optimize

BCL7C-specific considerations:

• Remember that BCL7C has a predicted molecular weight of 23 kDa

• Non-specific bands at 50 kDa and 25 kDa may represent IgG heavy and light chains

• Consider using loading controls and BCL7C-knockdown samples to identify specific bands

Validation approaches:

• Compare your results with the expected band pattern shown in reference blots from manufacturers

• Use multiple antibodies targeting different epitopes for confirmation

• Include appropriate controls (positive, negative, knockdown) in each experiment

How can I optimize immunohistochemistry protocols for BCL7C detection?

For optimal BCL7C detection in immunohistochemistry, implement these methodological refinements:

Tissue preparation and fixation:

• Standard 10% neutral buffered formalin fixation for 24-48 hours is generally appropriate

• Avoid overfixation which can mask epitopes

• For frozen sections, consider brief fixation in cold acetone or methanol

Antigen retrieval optimization:

• Test both heat-induced epitope retrieval methods:

  • Citrate buffer (pH 6.0) - try this first

  • EDTA buffer (pH 9.0) - alternative if citrate buffer gives weak signals

• Optimize retrieval time (typically 10-20 minutes)

• Consider pressure cooker-based retrieval for consistent results

Antibody incubation parameters:

• Titrate primary antibody concentration to find optimal dilution

• Test both short (1-2 hours room temperature) and long (overnight at 4°C) incubation times

• Use antibody diluent containing protein (BSA or serum) to reduce background

• Consider signal amplification systems for low-abundance targets

Controls:

• Include positive control tissues known to express BCL7C

• Employ negative controls (primary antibody omission and isotype controls)

• Consider dual staining with markers of specific cell types to confirm localization

Detection and counterstaining:

• Choose detection system based on required sensitivity (polymer-based systems often provide better signal-to-noise ratio)

• Optimize DAB development time for best signal-to-background ratio

• Use appropriate counterstain (hematoxylin) with optimal timing to prevent overcounterstaining

By systematically optimizing these parameters, you can achieve consistent and specific BCL7C staining for reliable immunohistochemical analysis.

What strategies can improve co-immunoprecipitation experiments with BCL7C antibodies?

To improve co-immunoprecipitation (co-IP) experiments investigating BCL7C interactions (particularly with mutant p53), consider these methodological strategies:

Lysis and buffer optimization:

• Use gentle lysis buffers (e.g., NP-40 or digitonin-based) to preserve protein interactions

• Based on the successful co-IP of BCL7C with mutant p53 variants, consider buffer conditions similar to those used in the study showing this interaction

• Include protease inhibitors to prevent degradation

• Perform lysis at 4°C to maintain protein complex integrity

Antibody selection and protocol:

• Choose antibodies specifically validated for immunoprecipitation

• Consider using tag-based systems (e.g., Myc-tagged BCL7C) if direct IP is challenging

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Optimize antibody amount through titration experiments

Washing and elution:

• Optimize wash stringency to balance removal of non-specific binding with preservation of specific interactions

• Consider graduated washing with buffers of increasing stringency

• Use appropriate elution conditions (competitive elution with peptides or direct boiling in SDS buffer)

Detection strategies:

• For BCL7C (23 kDa), be aware of potential overlap with antibody light chains (~25 kDa)

• Consider using antibody cross-linking to beads to prevent antibody co-elution

• Use antibodies from different host species for IP and blotting when possible

Controls for validation:

• Include IgG control immunoprecipitation

• Perform reciprocal co-IPs (e.g., IP with anti-BCL7C and blot for p53, then IP with anti-p53 and blot for BCL7C)

• Include input samples at different concentrations for quantitative assessment

These strategies can help optimize co-IP experiments investigating BCL7C's protein interactions, particularly its significant interaction with mutant p53 that underlies its tumor suppressor function .

What are the latest findings on BCL7C's role in cancer biology?

Recent research has revealed significant insights into BCL7C's role in cancer biology:

BCL7C as a tumor suppressor:

• High BCL7C expression correlates with favorable prognosis in ovarian cancer

• BCL7C suppresses proliferation of cancer cells harboring mutant p53

• Knockdown of BCL7C increases cancer cell proliferation and invasion

Mechanism of action:

• BCL7C physically interacts with multiple mutant p53 variants including mtp53-R175H, mtp53-Y220C, mtp53-R248W, mtp53-R249S, and mtp53-R273H

• This interaction leads to suppression of mutant p53 target gene expression

• The tumor-suppressive effect is specifically dependent on mutant p53, as demonstrated in experiments with p53-negative cell lines

Context-dependent effects:

• BCL7C shows tumor suppressive activity specifically in contexts where mutant p53 is present

• In p53-negative or wild-type p53 cells, BCL7C manipulation has minimal effects on proliferation

• This context-dependency suggests a specialized function in counteracting the oncogenic activities of mutant p53

Potential as a biomarker:

• BCL7C expression levels could potentially serve as a prognostic biomarker in cancers harboring mutant p53

• The combination of BCL7C expression and p53 status might provide more refined patient stratification

These findings establish BCL7C as a novel tumor suppressor that acts by counteracting the oncogenic activities of mutant p53, representing a significant advance in understanding how BCL7C contributes to cancer biology .

How can researchers study BCL7C in the context of chromatin remodeling complexes?

The association of BCL7C with chromatin remodeling processes requires specific methodological approaches:

Protein complex identification techniques:

• Co-immunoprecipitation with BCL7C antibodies followed by mass spectrometry

• Proximity-dependent biotinylation (BioID or TurboID) with BCL7C as bait

• Tandem affinity purification of tagged BCL7C to identify stable interaction partners

• Density gradient fractionation to isolate native BCL7C-containing complexes

Chromatin association studies:

• Chromatin immunoprecipitation (ChIP) using BCL7C antibodies to identify genomic binding sites

• ChIP-seq to map genome-wide BCL7C occupancy

• Re-ChIP (sequential ChIP) to identify co-occupancy with other chromatin remodeling factors

• CUT&RUN or CUT&Tag as alternatives to ChIP for mapping genomic interactions

Functional genomics approaches:

• RNA-seq following BCL7C knockdown or overexpression

• ATAC-seq to assess changes in chromatin accessibility

• HIGH-C to examine effects on chromatin organization

• CRISPRi/CRISPRa to modulate BCL7C expression and study chromatin effects

Microscopy-based methods:

• Immunofluorescence co-localization with known BAF complex components

• Proximity ligation assay to detect protein-protein interactions in situ

• Live-cell imaging with fluorescently tagged BCL7C to study dynamics

Since BCL7C is reported to be associated with the "BAF chromatin remodeling complex" , research should focus on interactions with core BAF components like BRG1/BRM, BAF155/170, and SNF5/INI1, and investigate how BCL7C contributes to the function of these complexes in regulating gene expression and chromatin structure.