bcl7ba Antibody

What is BCL7B and what is its significance in cancer research?

BCL7B is a member of the BCL7 family, which includes BCL7A, BCL7B, and BCL7C proteins. This family was first discovered when BCL7A was identified as being involved in complex translocation in a Burkitt lymphoma cell line . BCL7B specifically has emerged as an important biomarker in cancer research due to its differential expression across various cancer types and its association with patient prognosis.

Research has demonstrated that BCL7B expression has significant diagnostic and prognostic value in multiple cancer types. High BCL7B expression is associated with poor prognosis in glioblastoma multiforme (GBM), brain lower grade glioma (LGG), kidney chromophobe (KICH), oral squamous cell carcinoma (OSCC), rectum adenocarcinoma (READ), and uveal melanoma (UVM) . Conversely, low BCL7B expression correlates with poor outcomes in kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), skin cutaneous melanoma (SKCM), thyroid carcinoma (THCA), and sarcoma (SARC) .

Additionally, BCL7B has been associated with immune cell infiltration in tumor microenvironments, suggesting a potential role in tumor immunology and possible therapeutic implications .

What types of BCL7B antibodies are available for research applications?

Several types of BCL7B antibodies are available for different research applications. These include:

• Polyclonal antibodies: These recognize multiple epitopes on the BCL7B protein and are useful for detecting low abundance targets. Examples include rabbit polyclonal antibodies that react with human and mouse BCL7B .

• Monoclonal antibodies: These offer higher specificity by recognizing a single epitope. Available clones include 6D2 and 4G1 which target specific amino acid regions (AA 124-202) of BCL7B .

• Tagged and conjugated antibodies: Specialized BCL7B antibodies are available with various tags including biotin conjugates and fluorescent conjugates (e.g., AbBy Fluor® 647, AbBy Fluor® 350) .

What are the optimal sample preparation methods for BCL7B detection?

When preparing samples for BCL7B detection, researchers should consider the following methodological approaches:

For immunohistochemistry (IHC), optimal tissue fixation is crucial for preserving BCL7B epitopes. Formalin-fixed paraffin-embedded (FFPE) tissues are commonly used, with recommended antigen retrieval techniques to expose BCL7B epitopes that may be masked during fixation. For most BCL7B antibodies, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is recommended.

For western blotting applications, samples should be lysed in a buffer containing protease inhibitors to prevent protein degradation. The Human Protein Atlas database can be consulted for verifying BCL7B expression in different tissues before preparing samples . For optimal results, tissues known to have high BCL7B expression can serve as positive controls.

The recommended dilution range for BCL7B antibodies in IHC applications is typically 1:50-1:200, while for ELISA applications, a dilution range of 1:5000-1:10000 is suggested . These parameters should be optimized for each specific experimental setup.

What controls should be included when working with BCL7B antibodies?

Proper experimental controls are essential for validating BCL7B antibody results:

• Positive tissue controls: Include samples known to express BCL7B. According to research, tissues from adrenocortical carcinoma (ACC), cervical squamous cell carcinoma (CESC), and diffuse large B-cell lymphoma (DLBC) show high BCL7B expression and can serve as positive controls .

• Negative tissue controls: Include samples with low or no BCL7B expression. Tissues from bladder urothelial carcinoma (BCLA), colon adenocarcinoma (COAD), and thyroid carcinoma (THCA) demonstrate low BCL7B expression and can serve as negative controls .

• Isotype controls: Include an antibody of the same isotype (e.g., rabbit IgG) but not directed against BCL7B to control for non-specific binding.

• Antibody omission controls: Process samples without the primary antibody to assess background staining from secondary antibodies.

• Antibody absorption controls: Pre-incubate the BCL7B antibody with its specific immunogen (when available) to validate specificity.

How does BCL7B expression correlate with immune cell infiltration in different cancer types?

BCL7B expression shows significant correlations with the infiltration of 24 immune cell subsets across 37 different tumor environments, indicating its potential role in tumor immunology . Research using RNA-Seq expression profile data has revealed that BCL7B expression has varying degrees of correlation with immune cell infiltration patterns in different cancer types.

For instance, in adrenocortical carcinoma (ACC) and bladder urothelial carcinoma (BCLA), BCL7B expression shows distinct correlation patterns with different immune cell subtypes. The relationship between BCL7B expression and immune infiltration was analyzed using the GSVA R package (version 1.34.0) .

Additionally, Spearman's rank correlation coefficient analysis has demonstrated that BCL7B gene expression correlates with:

• 47 immune checkpoints

• 46 immune-activating genes

• 24 immune-suppressing genes

• 5 DNA repair genes

• DNA methylation, TMB, and MSI in 39 tumors

These findings suggest that BCL7B may influence the tumor immune microenvironment and potentially impact immunotherapy response, making it a valuable target for immunological research in oncology.

What are the best practices for validating BCL7B antibody specificity?

Validating antibody specificity is crucial for obtaining reliable results. For BCL7B antibodies, consider these methodological approaches:

• Western blot validation: Confirm the antibody detects a protein of the expected molecular weight (~55 kDa) in tissues known to express BCL7B. Compare results with publicly available data from the Human Protein Atlas .

• Knockout/knockdown controls: Use cells with CRISPR-mediated BCL7B knockout or siRNA-mediated knockdown to confirm antibody specificity.

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide (e.g., fusion protein of human BCL7B as used for some commercially available antibodies ) before application to samples. Signal reduction confirms specificity.

• Cross-reactivity testing: Test the antibody against other BCL7 family members (BCL7A, BCL7C) to ensure it doesn't cross-react, especially important when studying specific family member functions.

• Orthogonal validation: Compare results from multiple antibodies targeting different epitopes of BCL7B, or validate with non-antibody-based methods such as mRNA expression.

How can I use BCL7B antibodies to investigate its prognostic value in cancer research?

To investigate BCL7B's prognostic value in cancer research, consider these methodological approaches:

• Tissue microarray (TMA) analysis: Use validated BCL7B antibodies for immunohistochemical staining of TMAs containing samples from patients with known clinical outcomes. Score BCL7B expression levels (high vs. low) and correlate with survival data.

• Multiplex immunofluorescence: Combine BCL7B antibodies with markers for immune cell populations to simultaneously assess BCL7B expression and immune infiltration in the tumor microenvironment.

• Gene-protein correlation studies: Combine BCL7B protein expression data (from antibody-based methods) with BCL7B mRNA expression data to strengthen prognostic assessments.

• Survival analysis methodology: After BCL7B staining, use Kaplan-Meier survival analysis and Cox proportional hazards models to assess the relationship between BCL7B expression and patient outcomes.

Research has already demonstrated BCL7B's different prognostic implications across cancer types. For example, high BCL7B expression correlates with poor prognosis in glioblastoma multiforme but with better prognosis in kidney renal clear cell carcinoma . These contradictory patterns highlight the importance of cancer-specific analysis.

What approaches should be used to study BCL7B's interactions with other proteins?

To investigate BCL7B's protein-protein interactions, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): Use anti-BCL7B antibodies to pull down BCL7B and its interaction partners from cell lysates, followed by mass spectrometry or western blotting to identify bound proteins.

• Proximity ligation assay (PLA): This technique allows visualization of protein-protein interactions in situ. Use BCL7B antibodies in combination with antibodies against potential interaction partners.

• FRET/BRET analysis: For studying protein interactions in living cells, use fluorescent or bioluminescent tags in combination with BCL7B antibodies.

• Yeast two-hybrid screening: This can identify novel BCL7B interaction partners, which can then be validated using antibody-based methods.

• Chromatin immunoprecipitation (ChIP): If investigating BCL7B's potential role in transcriptional regulation, use BCL7B antibodies for ChIP assays followed by sequencing or PCR.

When designing these experiments, consider that BCL7B is part of the SWI/SNF complex involved in chromatin remodeling, which influences gene expression programs. Although not directly mentioned in the search results, this context is important for understanding potential interaction partners.

How do BCL7B expression patterns differ between normal and cancerous tissues?

Understanding differential expression patterns requires robust methodological approaches:

• Comparative immunohistochemistry: Use BCL7B antibodies to stain matched normal and tumor tissues from the same patients. The Human Protein Atlas database provides immunohistochemical staining images that can be used to verify BCL7B expression in normal tissues compared to tumor tissues .

• Quantitative analysis: Employ digital pathology and image analysis software to quantify BCL7B staining intensity and distribution in normal versus cancerous tissues.

• Subcellular localization analysis: Assess whether BCL7B protein exhibits altered subcellular localization in cancer cells compared to normal cells using high-resolution imaging.

Based on available research, BCL7B expression shows significant variation across cancer types. It is highly expressed in adrenocortical carcinoma (ACC), cervical squamous cell carcinoma (CESC), cholangiocarcinoma (CHOL), glioblastoma multiforme (GBM), and several other cancer types . Conversely, BCL7B expression is low in bladder urothelial carcinoma (BCLA), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), and others .

What are the optimal protocols for using BCL7B antibodies in Western blotting?

For optimal Western blotting results with BCL7B antibodies, consider the following methodological guidelines:

• Sample preparation: Prepare cell or tissue lysates using RIPA buffer supplemented with protease inhibitors. For tissues with low BCL7B expression, consider enrichment techniques such as immunoprecipitation before Western blotting.

• Gel electrophoresis parameters: Use 10-12% SDS-PAGE gels for optimal resolution of BCL7B protein, which has an expected molecular weight of approximately 55 kDa .

• Transfer conditions: Transfer proteins to PVDF membranes at 100V for 1 hour or 30V overnight at 4°C for best results.

• Blocking conditions: Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Antibody dilution: For most BCL7B antibodies, a dilution of 1:1000 is recommended for Western blotting applications .

• Detection method: Use HRP-conjugated secondary antibodies and enhanced chemiluminescence (ECL) for visualization. For low abundance detection, consider more sensitive substrates or amplification systems.

• Controls: Include positive controls (cell lines known to express BCL7B) and negative controls (BCL7B-null cell lines if available) to validate results.

What are the best practices for immunohistochemical detection of BCL7B?

For optimal immunohistochemical detection of BCL7B, follow these methodological guidelines:

• Tissue processing: Use formalin-fixed, paraffin-embedded (FFPE) tissues sectioned at 4-5 μm thickness.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) depending on the specific antibody requirements.

• Blocking: Block endogenous peroxidase activity with 3% hydrogen peroxide and non-specific binding with 5-10% normal serum.

• Antibody dilution: For IHC applications, most BCL7B antibodies work optimally at dilutions between 1:50 and 1:200 .

• Incubation conditions: Incubate primary antibody overnight at 4°C or for 1-2 hours at room temperature for best results.

• Detection system: Use a polymer-based detection system for enhanced sensitivity and reduced background.

• Counterstaining: Counterstain with hematoxylin to visualize tissue architecture and cellular context.

• Multiplex considerations: When performing multiplex IHC with BCL7B and other markers, carefully select antibodies from different host species or use specialized multiplex detection systems.

How can I troubleshoot weak or non-specific signals when using BCL7B antibodies?

When encountering issues with BCL7B antibody performance, consider these troubleshooting approaches:

• Weak or no signal issues:

  • Increase antibody concentration (try 2-5 fold higher concentration)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize antigen retrieval conditions (try different buffers or longer retrieval times)

  • Use a more sensitive detection system (amplification-based systems)

  • Check sample preparation and storage conditions

  • Verify BCL7B expression in your samples through alternative methods

• High background or non-specific signal issues:

  • Increase blocking time or concentration

  • Reduce primary antibody concentration

  • Reduce incubation time

  • Include additional washing steps

  • Use more specific secondary antibodies

  • Pre-absorb antibodies with relevant tissues

  • Use more stringent washing conditions (higher salt concentration)

• Cross-reactivity concerns:

  • Validate with knockout/knockdown controls

  • Perform peptide competition assays

  • Try alternative BCL7B antibodies targeting different epitopes

  • Use Western blotting to confirm specificity before IHC applications

• Tissue-specific optimization:

  • Different tissue types may require specific fixation and processing protocols

  • Consider tissue-specific antigen retrieval modifications

  • Adjust protease treatment for certain tissues if applicable

How can BCL7B antibodies be integrated into multiparameter flow cytometry panels?

For integrating BCL7B detection into flow cytometry applications, consider these methodological approaches:

• Antibody selection: Choose BCL7B antibodies conjugated to fluorophores that complement your existing panel. Options include BCL7B antibodies conjugated to AbBy Fluor® 647 or AbBy Fluor® 350 .

• Sample preparation: For intracellular BCL7B staining, use appropriate fixation and permeabilization reagents that preserve antigen recognition while allowing antibody access.

• Panel design considerations:

  • Account for spectral overlap between fluorophores

  • Include appropriate compensation controls

  • Consider BCL7B expression levels when selecting fluorophore brightness

  • Place BCL7B in appropriate fluorescence channel based on expected expression level

• Controls for flow cytometry:

  • Include FMO (fluorescence minus one) controls

  • Use isotype controls matched to BCL7B antibody

  • Include positive and negative cell controls for BCL7B expression

  • Consider using cells with manipulated BCL7B expression levels as additional controls

• Analysis strategies:

  • Use bivariate plots to correlate BCL7B expression with other markers

  • Consider dimensionality reduction techniques (tSNE, UMAP) for complex panels

  • Establish gating strategies based on controls and biological relevance

What approaches can be used to study BCL7B's role in immune infiltration?

To investigate BCL7B's relationship with immune infiltration, consider these methodological approaches:

• Multiplex immunohistochemistry/immunofluorescence:

  • Combine BCL7B antibodies with markers for specific immune cell subsets

  • Use multispectral imaging systems for simultaneous detection

  • Analyze spatial relationships between BCL7B-expressing cells and immune cells

• Single-cell analysis approaches:

  • Perform single-cell RNA sequencing (scRNA-seq) combined with protein analysis

  • Use CITE-seq or similar approaches to correlate BCL7B protein levels with transcriptional profiles

  • Analyze correlations between BCL7B expression and immune cell markers

• Computational analysis methods:

  • Apply the GSVA R package (version 1.34.0) as used in published research to analyze the relationship between BCL7B gene expression and immune cell infiltration

  • Use Spearman's rank correlation to assess relationships between BCL7B and immune checkpoints or immune-related genes

  • Perform GSEA (Gene Set Enrichment Analysis) to identify enriched immune-related pathways associated with BCL7B expression

• Functional validation experiments:

  • Manipulate BCL7B expression in tumor models and assess changes in immune infiltration

  • Perform co-culture experiments with immune cells and BCL7B-expressing or BCL7B-knockout tumor cells

  • Use conditional knockout models to study tissue-specific effects of BCL7B on immune responses