BCOR Antibody

What is BCOR protein and why is it important in research?

BCOR (BCL-6 interacting corepressor) is a 1,755 amino acid protein that functions as a transcriptional corepressor. It specifically inhibits gene expression when recruited to promoter regions by sequence-specific DNA-binding proteins such as BCL6 and MLLT3 . This repression may be mediated at least in part by histone deacetylase activities which can associate with this corepressor .

BCOR is involved in the repression of TFAP2A, impairing binding of BCL6 and KDM2B to TFAP2A promoter regions. Via repression of TFAP2A, it acts as a negative regulator of osteo-dentiogenic capacity in adult stem cells; this function implies inhibition of methylation on histone H3 'Lys-4' (H3K4me3) and 'Lys-36' (H3K36me2) .

What are the types of BCOR antibodies available for research?

Multiple types of BCOR antibodies are available for research purposes:

• Mouse Monoclonal Antibodies:

  • Clone C-10 (recognizes amino acids 1-300 at the N-terminus)

  • Clone BSB-128

• Rabbit Polyclonal Antibodies:

  • Targeting various epitopes including aa 150-350 and aa 1-100

  • Some with fusion protein immunogens

• Rabbit Monoclonal Antibodies:

  • Clone E6V3R (developed for ChIP, IP, and WB applications)

Each antibody type has specific applications and optimization requirements based on the particular experimental context and tissue type being studied.

What is the cellular localization of BCOR and how is this relevant for antibody selection?

BCOR primarily exhibits nuclear localization , which is crucial information when evaluating immunohistochemical staining results. When selecting a BCOR antibody, researchers should ensure that:

• The antibody demonstrates clear nuclear staining patterns

• Background cytoplasmic staining is minimal

• The nuclear staining pattern is consistent with the expected biology of BCOR

For accurate evaluation of BCOR immunohistochemistry, only nuclear staining should be counted as positive. Studies have established evaluation criteria where moderate to strong nuclear staining in more than 10% of tumor cells is considered positive .

How is BCOR antibody used in diagnosing small round cell tumors and other sarcomas?

BCOR antibody has emerged as a crucial diagnostic tool for small round cell tumors and various sarcomas:

• EWSR1-negative Small Blue Round Cell Tumors (SBRCTs):
  BCOR immunohistochemical staining is a highly sensitive marker for SBRCTs harboring specific genetic alterations .

• High-grade Endometrial Stromal Sarcomas:
  Strong diffuse nuclear BCOR staining (defined as >95% of tumor cells) was seen in the round cell component of 20 (100%) classic YWHAE-NUTM2 high-grade Endometrial Stromal Sarcomas and 3 unusual high-grade Endometrial Stromal Sarcomas .

• Clear Cell Sarcoma of the Kidney (CCSK):
  BCOR overexpression is common in CCSK cases, particularly those with BCOR genetic alterations, including BCOR rearrangement and internal tandem duplication .

• Pediatric Sarcomas:
  BCOR antibody helps identify a morphologic continuum of mixed round and spindle cell tumors in pediatric patients .

What is the significance of co-expression patterns with other markers in BCOR-positive tumors?

Research has shown important co-expression patterns that can enhance diagnostic accuracy when used alongside BCOR:

• BCOR with Cyclin D1: Studies show 93.7% positivity for Cyclin D1 in BCOR-positive sarcomas .

• BCOR with SATB2: 68.7% of BCOR-positive sarcomas demonstrate SATB2 positivity .

• BCOR with TLE1: Only 18.7% of BCOR-positive sarcomas show TLE1 positivity, making this a useful differential marker .

• BCOR with NKX2.2: NKX2.2 is consistently negative in BCOR-positive sarcomas, making it a helpful marker to differentiate Ewing sarcoma from sarcoma with BCOR genetic alterations .

• BCOR with CD99: CD99 is generally negative or shows non-specific cytoplasmic granular staining in BCOR-positive sarcomas, unlike Ewing sarcoma where CD99 often shows crisp, strong membranous staining .

How do genetic alterations correlate with BCOR protein expression?

Several genetic alterations have been associated with BCOR protein expression patterns:

• BCOR Internal Tandem Duplications (ITD): Commonly seen in Clear Cell Sarcoma of the Kidney and associated with BCOR overexpression .

• YWHAE-NUTM2 fusions: High-grade endometrial stromal sarcomas with this fusion show strong, diffuse BCOR immunopositivity in 100% of cases .

• ZC3H7B-BCOR/BCOR-ZC3H7B fusions: In high-grade endometrial stromal sarcomas, these fusions show variable BCOR expression patterns, with strong expression in some cases and weak or absent expression in others .

• BCOR-CCNB3 and BCOR-MAML3 fusions: These fusions are observed in small blue round cell tumors that demonstrate BCOR overexpression by immunohistochemistry .

The molecular diagnosis of these various BCOR genetic alterations requires elaborate methodology including custom BAC FISH probes and RT-PCR assays, making immunohistochemistry a valuable surrogate marker for initial screening .

What are the optimal protocols for BCOR immunohistochemistry?

Based on the literature, the following protocols have yielded reliable results:

• Antibody Selection:

  • Clone C-10 (sc-514576; Santa Cruz) diluted at 1:150 (1.7μg/ml) has shown robust results

  • BSB-128 clone has also demonstrated good performance in diagnostic settings

• Antigen Retrieval:

  • Heat-based antigen retrieval using high pH buffer solution (Leica, ER2, 30 minutes)

  • Alternative methods include citrate buffer pH 6.0

• Primary Antibody Incubation:

  • 30 minutes at room temperature shows optimal results

• Detection System:

  • Polymer-based secondary systems (e.g., Refine, Leica) yield clean backgrounds

• Evaluation Criteria:

  • Moderate to strong nuclear staining in >10% of tumor cells is considered positive

  • For high-grade endometrial stromal sarcomas, diffuse staining in >95% of tumor cells is diagnostic

What controls should be used when validating BCOR antibody performance?

Appropriate controls are essential for validating BCOR antibody performance:

• Positive Tissue Controls:

  • Testis (particularly spermatogonia in seminiferous tubules)

  • Endometrial stromal sarcoma (molecularly confirmed YWHAE-NUTM2 positive)

  • Clear cell sarcoma of the kidney

• Negative Tissue Controls:

  • Multi-tissue blocks containing normal tissues (spleen, placenta, kidney, colon, liver, skin, pancreas, and lung)

  • Uterine leiomyomas (consistently BCOR-negative)

• Cell Line Controls:

  • HEK-293 cells, HeLa cells, and K-562 cells have been validated for western blot applications

How can researchers troubleshoot weak or false-negative BCOR staining?

When encountering weak or absent BCOR staining, consider the following troubleshooting steps:

• Fixation Issues:

  • Overfixation or underfixation can affect antigen retrieval efficiency

  • Standardize fixation time (24-48 hours in 10% neutral buffered formalin is recommended)

• Antigen Retrieval Optimization:

  • Extend antigen retrieval time (try 40-45 minutes instead of 30)

  • Test alternative pH buffers (high pH vs. citrate buffer)

• Antibody Concentration:

  • Titrate antibody concentration (try 1:100 for C-10 clone if 1:150 yields weak results)

• Incubation Time:

  • Extend primary antibody incubation (try overnight at 4°C if 30 minutes at room temperature is insufficient)

• Detection System:

  • Use amplification systems for weak signals

  • Consider tyramide signal amplification for difficult cases

• Storage Considerations:

  • Antibody degradation can occur; store vials at 2-8°C (most BCOR antibodies are stable for 24 months under these conditions)

How does BCOR antibody performance differ across various applications?

BCOR antibodies have been validated across multiple applications with varying performance characteristics:

For western blot applications, researchers should note that BCOR appears at approximately 186-192 kDa, with some antibodies also detecting a lower molecular weight band around 110 kDa that may represent an alternative isoform or cleavage product .

How does BCOR antibody staining pattern differ between pediatric and adult sarcomas?

Research on age-related differences in BCOR expression has revealed important distinctions:

• Pediatric BCOR-positive Sarcomas:

  • Average age: 5.94 ± 5.2 years, significantly younger than BCOR-negative cases (9.39 ± 5.22 years)

  • Often present with distinctive histologic features including:

    • Both spindle and round cell components (87.5%)

    • Alternating hypo- and hypercellular areas (75%)

    • Prominent myxoid stroma (50%)

    • Rich and branching vasculature (63%)

• Adult BCOR-positive Sarcomas:

  • More commonly associated with endometrial stromal sarcomas in women

  • Often show strong, diffuse BCOR expression in high-grade tumors (particularly YWHAE-NUTM2 positive cases)

A statistically significant difference has been observed between the histological features of BCOR-positive and BCOR-negative tumors, with BCOR-positive cases frequently showing a distinct morphologic pattern that can aid in identifying cases for molecular testing .

What is the relationship between BCOR protein expression and clinical outcomes?

Studies investigating the correlation between BCOR expression and patient outcomes have found:

How does BCOR antibody usage vary between research and diagnostic applications?

Important distinctions exist between research and diagnostic applications:

• Research Applications:

  • Multiple antibody formats may be acceptable (polyclonal, monoclonal)

  • Broader range of applications (WB, IP, ChIP, IF/ICC)

  • Focus on mechanism elucidation and molecular interactions

  • May require custom protocols and optimization

• Diagnostic Applications:

  • IVD-certified antibodies are required for clinical use

  • Standardized protocols are essential for reproducibility

  • Interpretation requires qualified pathologists

  • Results must be integrated with clinical history and other diagnostic tests

  • Performance characteristics must be validated in specific tumor types

For diagnostic use, BCOR (C-10) Mouse Monoclonal Primary Antibody is intended for laboratory professional use in the detection of the BCOR protein in formalin-fixed, paraffin-embedded tissue stained in manual qualitative immunohistochemistry testing, to be used after the primary diagnosis of tumor has been made by conventional histopathology .

What emerging applications of BCOR antibodies are being explored?

Several innovative applications are currently being developed:

• Liquid Biopsy Biomarkers:

  • Detection of circulating BCOR protein in blood samples from patients with BCOR-altered tumors

  • Potential for non-invasive monitoring of disease progression

• Therapeutic Target Identification:

  • Using BCOR antibodies to identify patients who might benefit from targeted therapies against BCOR-related pathways

  • Investigating BCOR's role in treatment resistance mechanisms

• Multiplexed Immunohistochemistry:

  • Combining BCOR with other markers in multiplexed panels for improved diagnostic accuracy

  • Integration with digital pathology and AI-based image analysis

• Single-Cell Analysis:

  • Applications in single-cell proteomics to understand heterogeneity in BCOR expression within tumors

  • Correlation with single-cell transcriptomics data

How can researchers validate novel BCOR antibodies for specialized applications?

When validating new BCOR antibodies for specialized applications, consider:

• Epitope Mapping:

  • Determine the specific regions of BCOR recognized by the antibody

  • Consider potential cross-reactivity with similar proteins or isoforms

• Knockout/Knockdown Validation:

  • Use CRISPR-Cas9 or siRNA to create BCOR-depleted controls

  • Confirm specificity by demonstrating loss of signal in BCOR-depleted samples

• Orthogonal Method Validation:

  • Compare antibody results with RNA-seq or proteomics data

  • Validate findings across multiple platforms and techniques

• Multiple Antibody Comparison:

  • Test new antibodies alongside established antibodies like C-10 clone

  • Compare staining patterns, sensitivity, and specificity

• Molecular Correlation:

  • Correlate antibody staining with molecular testing for BCOR alterations

  • Establish sensitivity and specificity for detecting various BCOR genetic events