CBFA2T2 Antibody

What is CBFA2T2 and what cellular processes is it involved in?

CBFA2T2 (Core-Binding Factor Subunit Alpha-2-Like 2) is a transcriptional co-repressor protein that plays crucial roles in several biological processes:

• Functions as a transcriptional regulator in hematopoiesis and skeletal development

• Serves as a novel co-repressor protein regulating pluripotency and germline specification

• Forms biochemical complexes with transcription factors such as PRDM14 and OCT4

• Oligomerizes to form a scaffold that stabilizes transcription factors on chromatin

• Involved in the regulation of PGC (Primordial Germ Cell) specification and maturation

CBFA2T2 is significant in both normal development and disease states, particularly in leukemia where it can bind to AML1-MTG8 fusion protein in acute myeloid leukemia .

What are the common synonyms and alternative names for CBFA2T2?

CBFA2T2 is known by several alternative names in the literature:

These synonyms are important to recognize when searching literature or ordering antibodies, as different manufacturers may use different nomenclature .

What are the key applications for CBFA2T2 antibodies in research?

CBFA2T2 antibodies have been validated for several experimental applications:

These applications allow researchers to investigate CBFA2T2 expression, localization, and interactions in various experimental contexts .

What cell lines and tissue samples serve as positive controls for CBFA2T2 antibody validation?

Reliable positive controls are essential for antibody validation. For CBFA2T2, the following samples have demonstrated positive reactivity:

Cell Lines:

• Jurkat (human T lymphocyte)

• Raji (human B lymphocyte)

• HepG2 (human liver cancer)

• SW620 (human colorectal adenocarcinoma)

• HEK-293 (human embryonic kidney)

• HeLa (human cervical cancer)

Tissue Samples:

• Human testis tissue

• Mouse testis tissue

• Human gliomas tissue

When validating a new CBFA2T2 antibody, these samples provide reliable positive controls, though expression levels may vary by sample type and preparation method .

What are the optimal protocols for using CBFA2T2 antibodies in Western blot applications?

For optimal Western blot results with CBFA2T2 antibodies, the following methodological considerations are recommended:

• Sample Preparation:

  • Use cell lysates from positive control cell lines (HEK-293, HeLa, Jurkat)

  • Include mouse testis tissue as a positive control for animal studies

• Gel Electrophoresis:

  • Load adequate protein (20-50 μg per lane)

  • CBFA2T2 has a calculated molecular weight of 67 kDa but is observed at approximately 80 kDa due to post-translational modifications

• Antibody Dilution:

  • Primary antibody dilution: 1:500 - 1:2000 (optimize for each antibody lot)

  • Incubation: Overnight at 4°C for best results

• Detection:

  • Use secondary antibody specific to rabbit IgG (as most CBFA2T2 antibodies are rabbit polyclonal)

  • Expected band: ~80 kDa

This approach maximizes detection sensitivity while minimizing background signal in Western blot applications.

How should researchers address cross-reactivity concerns with CBFA2T2 antibodies?

Cross-reactivity is a critical concern when working with antibodies against CBFA2T2, particularly due to its homology with other family members. To address this:

• Validate specificity through multiple approaches:

  • Compare reactivity patterns across different cell lines with known CBFA2T2 expression profiles

  • Include knockout or knockdown controls when possible

  • Consider that CBFA2T2 has 85% sequence similarity with other homologs (ETO and CBFA2T3)

• Apply orthogonal validation:

  • Confirm protein detection with antibodies targeting different epitopes

  • Correlate protein detection with RNA expression data (RNA-seq) as seen in enhanced validation approaches

• Test reactivity in multiple species:

  • Verify specificity across human, mouse, and rat samples if cross-species reactivity is claimed

These approaches help distinguish between specific CBFA2T2 detection and potential cross-reactivity with other family members or non-specific binding.

How does CBFA2T2 function in the regulation of pluripotency and germline specification?

CBFA2T2 plays a sophisticated role in pluripotency and germline specification through several molecular mechanisms:

• Complex formation with key transcription factors:

  • CBFA2T2 forms a biochemical complex with PRDM14, a germline-specific transcription factor

  • This complex co-localizes with OCT4, SOX2, and NANOG (OSN) at regulatory elements across the genome

• Scaffolding function:

  • CBFA2T2 oligomerizes through its NHR2 domain to form a large molecular scaffold

  • This scaffold stabilizes PRDM14 and OCT4 on chromatin at their target sites

  • Unlike traditional "passenger" co-repressors, CBFA2T2 actively promotes stable TF binding by limiting "off-rate" from chromatin

• Transcriptional control:

  • CBFA2T2/PRDM14 co-targeted genes include numerous transcription factors involved in lineage commitment

  • In embryonic stem cells, CBFA2T2 helps maintain pluripotency by preventing premature lineage commitment

The molecular evidence comes from ChIP-seq data showing 1,384 genomic regions where CBFA2T2 and PRDM14 binding events overlap, with 1022 co-targeted genes identified .

What phenotypes are observed in CBFA2T2 knockout models?

CBFA2T2 knockout models display striking developmental phenotypes, particularly in germline development:

• Cellular phenotypes in vitro:

  • Cbfa2t2 knockout mESCs display flattened morphology

  • These cells cannot be maintained without kinase inhibitors of MAPK/ERK and GSK3 (2i conditions)

  • RNA-seq analysis shows dysregulation of numerous pluripotency genes (downregulated: Klf4, Pou5f1, Nr0b1, Lin28a, Myc) and upregulation of lineage commitment genes

• Germline development in vivo:

  • Cbfa2t2−/− mice exhibit severe germline defects

  • Female mice have underdeveloped ovaries with complete absence of follicles

  • Male mice have testes reduced to ~30% of wild-type size

  • Sperm count reduced to less than 10% of wild-type with remaining sperm largely immotile

  • Alkaline phosphatase staining shows >95% reduction in PGC numbers in the genital ridge of E11.5 embryos

  • Defects occur as early as E7.25-E8.75 in embryonic development

• Viability:

  • Despite germline defects, Cbfa2t2−/− mice are viable and born in roughly normal Mendelian ratios

These findings establish CBFA2T2 as an essential factor for germline development, particularly in PGC specification and maturation.

What is the molecular mechanism by which CBFA2T2 oligomerization stabilizes transcription factors on chromatin?

The mechanism of CBFA2T2-mediated stabilization of transcription factors represents a paradigm shift in understanding co-repressor function:

This molecular mechanism challenges the traditional view of co-repressors as passive factors in transcription factor recruitment, positioning CBFA2T2 as an active scaffold essential for stable transcription factor binding.

What is the relationship between CBFA2T2 and acute myeloid leukemia pathogenesis?

CBFA2T2 has significant implications in leukemia pathogenesis through several mechanisms:

• Interaction with leukemia-associated fusion proteins:

  • CBFA2T2 (also known as MTGR1) can bind to the AML1-MTG8 fusion protein in acute myeloid leukemia

  • Overexpression of MTGR1 can stimulate AML1-MTG8 to induce GCSF-dependent proliferation of L-G cells

  • MTGR1 can interfere with AML1-dependent transcription when overexpressed

• Complex formation:

  • AML1/MTG8 functions as a complex with MTGR1

  • This complex may play an important role in promoting leukemogenesis

• Deletion in leukemia:

  • MTGR1 (CBFA2T2) is found deleted in some cases of acute myeloid leukemia

  • This suggests complex roles that may be context-dependent

Understanding these interactions provides insights into potential therapeutic targets in leukemia treatment and highlights the context-dependent functions of CBFA2T2 in normal development versus disease states.

How can researchers optimize immunohistochemistry protocols for CBFA2T2 detection?

For successful IHC detection of CBFA2T2, consider these methodological refinements:

• Antigen retrieval optimization:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Heat-induced epitope retrieval (HIER) methods typically yield better results than proteolytic methods

• Antibody dilution titration:

  • Start with manufacturer's recommended range (typically 1:20-1:200)

  • Perform serial dilutions to determine optimal concentration for your specific tissue

  • Sample-dependent optimization is critical for successful detection

• Positive control tissue selection:

  • Human testis tissue shows reliable CBFA2T2 expression

  • Human gliomas tissue has also demonstrated positive reactivity

  • Include these as positive controls alongside your experimental tissues

• Signal amplification considerations:

  • For low-abundance expression, consider using polymer-based detection systems

  • Longer primary antibody incubation (overnight at 4°C) may improve detection of low-abundance targets

These optimizations should be systematically tested to achieve specific CBFA2T2 detection while minimizing background in your particular tissue samples.

What approaches can resolve discrepancies between protein and RNA expression data for CBFA2T2?

When protein detection by antibodies doesn't match RNA expression data, consider these investigative approaches:

• Validate antibody specificity:

  • Test multiple antibodies targeting different epitopes of CBFA2T2

  • Include knockout/knockdown controls to confirm specificity

  • Check for post-translational modifications that might affect epitope recognition

• Consider protein stability and turnover:

  • CBFA2T2 might undergo tissue-specific post-translational regulation

  • Proteasomal degradation rates may differ between tissues

  • Use proteasome inhibitors to assess protein stability

• Examine alternative splicing:

  • Verify whether the antibody epitope is present in all splice variants

  • The CBFA2T2 sequence used for immunogen development typically corresponds to amino acids 325-604 of human CBFA2T2 (NP_005084.1)

• Correlation with orthogonal methods:

  • Compare immunohistochemistry results with corresponding RNA-seq data

  • Use enhanced validation approaches like those employed by Atlas Antibodies that correlate protein detection with RNA expression patterns

This systematic approach helps determine whether discrepancies represent biological reality or technical limitations in either protein or RNA detection methods.