CBFB Antibody

What is CBFB and what is its molecular function?

CBFB forms the heterodimeric complex core-binding factor (CBF) with RUNX family proteins (RUNX1, RUNX2, and RUNX3). While CBFB does not bind DNA directly, it functions as a non-DNA-binding regulatory subunit that allosterically enhances the sequence-specific DNA-binding capacity of RUNX proteins . The heterodimers recognize the core consensus binding sequence 5'-TGTGGT-3', or rarely 5'-TGCGGT-3', within regulatory regions of target genes . These complexes bind to core sites of various enhancers and promoters, including murine leukemia virus, polyomavirus enhancer, T-cell receptor enhancers, and promoters of genes like LCK, IL3, and GM-CSF . CBFB complexes also play a role in T cell development by repressing the ZBTB7B transcription factor during cytotoxic (CD8+) T cell differentiation .

A comprehensive validation strategy for CBFB antibodies should include:

• Positive controls: Use cell lines known to express CBFB, such as Jurkat, HEK-293, MOLT-4, K-562, or HSC-T6 cells .

• Negative controls: Implement CBFB knockdown using validated shRNA or siRNA sequences. For example, shRNA clones V2LHS-89195 (A6) and V3LHS-639151 (B10) have been effectively used to silence CBFB expression .

• Western blot validation: Confirm detection of a single specific band at approximately 22 kDa, which is the calculated molecular weight of CBFB .

• Cross-reactivity testing: If working with multiple species, verify antibody reactivity in each target species separately.

• Application-specific controls: For ChIP applications, include IgG controls and input samples ; for immunofluorescence, include secondary-only controls and CBFB-depleted samples .

What are the optimal storage conditions and handling recommendations for CBFB antibodies?

For long-term stability and performance, CBFB antibodies should be:

• Stored at -20°C, where they remain stable for up to one year after shipment .

• Prepared in appropriate storage buffers, such as PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Aliquoted to avoid repeated freeze-thaw cycles, although some formulations may not require aliquoting for -20°C storage .

• Small volume preparations (e.g., 20μl sizes) may contain 0.1% BSA as a stabilizer .

• Always handle antibodies according to the manufacturer's specific recommendations, as formulations may vary.

How should Western blot protocols be optimized for CBFB detection?

For optimal CBFB detection by Western blot:

• Sample preparation: Extract proteins using RIPA or NP-40 based lysis buffers with protease inhibitors.

• Protein loading: Load 20-50 μg of total protein per lane.

• Gel selection: Use 10-12% SDS-PAGE gels for optimal resolution around the 22 kDa range.

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

• Primary antibody: Dilute according to manufacturer's recommendations, typically 1:1000-1:50000 . Incubate overnight at 4°C.

• Secondary antibody: Use appropriate HRP-conjugated secondary (e.g., anti-rabbit for most CBFB antibodies).

• Detection: Visualize using enhanced chemiluminescence (ECL) substrates.

• Controls: Include positive control lysates from Jurkat, HEK-293, MOLT-4, K-562, or HSC-T6 cells .

• Expected result: A specific band should be detected at approximately 22 kDa .

What are the recommended protocols for immunoprecipitation of CBFB?

For effective immunoprecipitation of CBFB:

• Lysis buffer: Use non-denaturing lysis buffer (20 mM Tris HCl pH 8, 137 mM NaCl, 1% Nonidet P-40, 2 mM EDTA) with freshly added protease inhibitors .

• Sample preparation: Place cell culture dish on ice, wash cells with ice-cold PBS, then add ice-cold lysis buffer .

• Antibody incubation: Mix specific CBFB antibody with the sample to capture the target protein.

• Immunoprecipitation: Add Protein A or G affinity chromatography colloid or magnetic beads to form an "antigen-antibody-colloid" complex .

• Co-immunoprecipitation: For studying CBFB-RUNX interactions, ensure complete cell lysis under non-denaturing conditions to preserve protein-protein interactions .

• Controls: Include IgG controls and input samples to assess specificity and efficiency.

How can immunofluorescence protocols be optimized for CBFB visualization?

For optimal CBFB immunofluorescence staining:

• Fixation: Fix cells with 4% paraformaldehyde for 15-20 minutes at room temperature .

• Permeabilization: Permeabilize with an appropriate detergent like Triton X-100.

• Blocking: Block with normal serum matching the secondary antibody host species.

• Primary antibody: Apply CBFB antibody at the recommended dilution (typically 1:50-1:200) and incubate overnight at 4°C or for 3+ hours at room temperature.

• Secondary antibody: Use appropriate fluorophore-conjugated secondary antibody (e.g., NorthernLights™ 557-conjugated anti-sheep IgG has been successfully used) .

• Nuclear counterstain: Apply DAPI for nuclear visualization .

• Mounting: Use anti-fade mounting medium to preserve fluorescence.

• Expected results: CBFB staining should be observed primarily in the nucleus, though cytoplasmic staining may also be present in some cell types .

What are the considerations for using CBFB antibodies in ChIP and ChIP-seq experiments?

For successful ChIP/ChIP-seq with CBFB antibodies:

• Antibody selection: Use ChIP-grade antibodies specifically validated for this application, such as ab195411 .

• Crosslinking: Optimize formaldehyde concentration and crosslinking time for your specific cell type.

• Sonication: Adjust sonication parameters to generate DNA fragments of appropriate size (200-500 bp).

• Immunoprecipitation: Use sufficient antibody amounts and appropriate bead selection.

• Controls: Include IgG control immunoprecipitations and input samples.

• Data analysis: Since CBFB does not directly bind DNA, analyze in conjunction with RUNX ChIP-seq data to identify co-occupied regions.

• Target sequences: Focus analysis on regions containing the RUNX consensus binding sequence (5'-TGTGGT-3' or 5'-TGCGGT-3') .

How can I study CBFB-RUNX interactions in cellular models?

To investigate CBFB-RUNX interactions:

• Co-immunoprecipitation: Use non-denaturing lysis conditions to maintain native protein complexes. Immunoprecipitate with CBFB antibody and blot for RUNX proteins, or vice versa .

• Proximity ligation assay (PLA): Detect in situ protein-protein interactions with high sensitivity using antibodies against CBFB and RUNX proteins.

• Fluorescence co-localization: Perform dual immunofluorescence with antibodies against CBFB and RUNX proteins to visualize subcellular co-localization patterns.

• Functional studies: Assess the effect of CBFB knockdown or overexpression on RUNX-dependent transcriptional activity.

• Mutational analysis: Create CBFB mutants and assess their ability to interact with RUNX proteins to map interaction domains.

Research has shown that the CBFB-MYH11 fusion protein sequesters RUNX1 in the cytoplasm, preventing its normal nuclear function, which can be visualized using appropriate antibodies .

How can I detect and study the CBFB-MYH11 fusion protein in leukemia research?

For investigating CBFB-MYH11 fusion in acute myeloid leukemia:

• Western blot: Use antibodies targeting the N-terminus of CBFB to detect both wild-type CBFB (22 kDa) and the fusion protein (higher molecular weight).

• Immunofluorescence: Examine subcellular localization changes, as CBFB-MYH11 sequesters RUNX1 in the cytoplasm .

• T-cell recognition assays: As demonstrated in research, CBFB-MYH11 can serve as a neoantigen enabling T cell recognition and killing of AML cells .

• Patient-derived xenograft models: Use CBFB antibodies to monitor fusion protein expression in PDX models, such as the MISTRG mice that reliably engraft with CBFB-MYH11+ AML .

• Functional studies: Investigate how CBFB-MYH11 alters gene expression compared to wild-type CBFB, as research has shown it affects expression of genes also regulated by DNMT3A .

How can CBFB antibodies be used to study epigenetic regulation?

For investigating CBFB's role in epigenetic regulation:

• ChIP-seq for CBFB and histone marks: Perform parallel ChIP-seq for CBFB and relevant histone modifications to correlate binding with chromatin states.

• DNA methylation analysis: Use methylated DNA immunoprecipitation (MeDIP-qPCR) to study methylation changes at CBFB target genes, as demonstrated in studies showing CBFB-MYH11 expression reduces 5mC levels of genes including GATA6, SPHK1, and JUN .

• Expression correlation: Compare CBFB binding patterns with gene expression changes, as research has identified overlapping sets of upregulated genes in both CBFB-MYH11 expressing cells and DNMT3A knockdown cells .

• Protein interactions: Investigate whether CBFB interacts with epigenetic modifiers. Research has shown that CBFB-MYH11 does not directly interact with DNMT3A, DNMT3B, or DNMT3L, but affects methylation through sequestering RUNX1 in the cytoplasm .

What approaches can be used to study CBFB's role in RNA binding?

Based on research indicating CBFB binds to RNA transcripts:

• RNA immunoprecipitation (RIP): Perform RIP followed by deep sequencing (RIP-seq) to identify CBFB-bound transcripts. Research has identified 837 CBFB-bound transcripts with fold enrichment > 4 .

• Validation by RT-PCR: Confirm binding of CBFB to specific transcripts of interest using RT-PCR on immunoprecipitated RNA.

• Comparative analysis: Compare CBFB-bound transcripts with those bound by other RNA-binding proteins. Research found 90% of CBFB-bound transcripts were also bound by hnRNPK .

• Functional studies: Investigate how CBFB binding affects RNA stability, processing, or translation of target transcripts.

• Motif analysis: Identify common sequence or structural motifs in CBFB-bound RNAs.

How can CBFB antibodies be used to study osteoarthritis?

Recent research has implicated CBFB in osteoarthritis (OA) pathogenesis:

• Expression analysis: Compare CBFB expression in normal and OA cartilage using immunohistochemistry or Western blot. Research has shown decreased expression of CBFB in cartilage of human OA patients .

• Methylation studies: Examine methylation at the CBFB promoter, as Methyl-seq data has revealed increased methylation in OA patient hip tissue compared to healthy individuals .

• Signaling pathway analysis: Investigate how CBFB modulates Wnt/β-catenin, Hippo/Yap, and Tgfβ signaling pathways in articular cartilage .

• Animal models: Use CBFB antibodies to study expression in chondrocyte-specific knockout models (e.g., Cbfb f/f;Col2a1-CreERT mice) or surgical OA models (ACLT or DMM) .

• Therapeutic studies: Monitor CBFB levels following AAV-mediated Cbfb overexpression, which has shown protective effects against OA in mouse models .

What are the applications of CBFB antibodies in cancer research beyond leukemia?

CBFB antibodies have important applications in various cancer contexts:

• Breast cancer research: CBFB is frequently mutated in breast cancer and plays a significant role in cancer pathogenesis . Use immunohistochemistry to examine CBFB expression patterns in breast cancer tissues.

• Transcriptional regulation: Investigate how CBFB regulates gene expression in cancer cells, as it forms complexes with RUNX proteins to modulate transcription of target genes .

• Cancer-associated exosomes: Study CBFB in cancer-associated exosomes, which may facilitate aggressive behavior in cancer .

• RNA binding: Explore CBFB's role in suppressing cancer through RNA binding mechanisms .

• Therapeutic target validation: Use CBFB antibodies to confirm target engagement in preclinical studies of therapies directed at CBFB pathways.

How can CBFB antibodies be used in immunotherapy research?

Based on emerging research in cancer immunotherapy:

• Neoantigen identification: CBFB-MYH11 fusion proteins can serve as neoantigens that enable T cell recognition and killing of AML cells .

• T cell response assessment: Use CBFB antibodies to confirm expression of fusion proteins in target cells when evaluating T cell responses.

• Patient-derived xenograft models: Monitor fusion protein expression in PDX models used for testing immunotherapeutic approaches .

• TCR T cell development: Support the development of T cell receptor (TCR) T cell immunotherapy targeting fusion gene-driven AML .

• Treatment monitoring: Assess changes in CBFB or fusion protein expression during immunotherapy treatment.

Research has demonstrated that high-avidity CBFB-MYH11 epitope-specific T cell receptors transduced into CD8+ T cells conferred antileukemic activity in vitro, providing proof of principle for targeting AML-initiating fusions immunologically .

What are common technical issues when working with CBFB antibodies and how can they be addressed?

Researchers may encounter several challenges when working with CBFB antibodies:

• Non-specific binding: Optimize blocking conditions and antibody dilutions. Validate specificity using CBFB knockdown controls.

• Weak signal in Western blot: Increase protein loading, optimize antibody concentration, or use more sensitive detection methods. Consider using validated positive control samples like Jurkat, HEK-293, or K-562 cell lysates .

• Background in immunofluorescence: Improve blocking, reduce primary antibody concentration, increase washing steps, or use a different secondary antibody.

• ChIP efficiency issues: Optimize crosslinking conditions, sonication parameters, and antibody amounts. Validate the antibody specifically for ChIP applications .

• Variable results between experiments: Standardize protocols, use the same antibody lot when possible, and include consistent positive and negative controls.

How do I select the most appropriate CBFB antibody for my specific research question?

Consider these factors when selecting a CBFB antibody:

• Application compatibility: Choose antibodies validated for your specific application (WB, IP, ChIP, IF, FC) .

• Species reactivity: Verify the antibody reacts with your species of interest. Some antibodies react with human, mouse, and rat samples .

• Epitope location: Select antibodies targeting appropriate regions of CBFB based on your research questions. For detecting both wild-type and fusion proteins, choose antibodies against conserved regions.

• Clonality: Monoclonal antibodies offer high specificity but limited epitope recognition; polyclonal antibodies provide broader epitope recognition but potential batch variation.

• Validation data: Review published literature and manufacturer data showing successful use of the antibody in similar experimental contexts.

• Citation record: Consider antibodies with established track records in peer-reviewed publications .

What controls are essential when studying CBFB through knockdown or overexpression?

Implement these critical controls in CBFB modulation experiments:

• Multiple siRNA/shRNA sequences: Use at least two different validated sequences targeting CBFB to rule out off-target effects. Research has successfully used shRNA clones V2LHS-89195 and V3LHS-639151 .

• Scrambled/non-targeting controls: Include appropriate negative controls for knockdown experiments.

• Empty vector controls: For overexpression studies, include the empty vector (e.g., empty pLVX vector) .

• Expression verification: Confirm CBFB knockdown or overexpression by Western blot using validated antibodies .

• Functional validation: Verify the impact on known CBFB-regulated genes or processes, such as effects on RUNX-dependent transcription.

• Rescue experiments: Restore CBFB expression with an RNAi-resistant construct to confirm specificity of observed phenotypes.