CREBBP Antibody

What are the most common applications for CREBBP antibodies in research?

CREBBP antibodies are primarily utilized in a variety of experimental applications including:

• Western Blot (WB): Typically used at dilutions of 1:1000-1:4000, effective for detecting the 265-290 kDa CREBBP protein

• Immunohistochemistry (IHC-P): Generally used at dilutions of 1:50-1:600

• Immunofluorescence/Immunocytochemistry (IF/ICC): Recommended at dilutions of 1:100-1:1600

• Immunoprecipitation (IP): Particularly useful for protein-protein interaction studies

• Flow Cytometry (FC): For intracellular detection at approximately 0.25 µg per 10^6 cells

For optimal results across applications, it is recommended that researchers titrate the antibody concentration in each testing system to determine ideal conditions for specific experimental setups.

How should I prepare samples for detecting CREBBP in cells by immunofluorescence?

For successful immunofluorescence detection of CREBBP in cultured cells, follow this validated protocol:

• Seed cells onto appropriate surfaces (poly-L-lysine coated glass slides for suspension cells)

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Wash three times with PBS (5 minutes per wash)

• Permeabilize with 0.1% Triton X-100 for 5-10 minutes

• Block with 2-3% BSA in PBS for 30-60 minutes at room temperature

• Incubate with primary CREBBP antibody (diluted 1:100-1:400 in blocking solution) overnight at 4°C

• Wash three times with PBS

• Incubate with appropriate fluorophore-conjugated secondary antibody

• Counterstain nuclei with DAPI

• Mount and visualize

This protocol has been validated for cell lines including HeLa, NIH-3T3, and leukemia cells, demonstrating the nuclear localization pattern characteristic of CREBBP.

What is the expected molecular weight for CREBBP detection in Western blotting?

When performing Western blot analysis for CREBBP:

The discrepancy between calculated (265 kDa) and observed (290-300 kDa) molecular weights is common for CREBBP and likely results from post-translational modifications. When troubleshooting, ensure adequate protein transfer of high-molecular-weight proteins by using appropriate transfer conditions and confirming with reversible protein stains .

How can I optimize CREBBP antibody-based co-immunoprecipitation for studying protein-protein interactions?

For effective co-immunoprecipitation of CREBBP and its interacting partners:

• Cell Lysis: Use Co-RIPA buffer or equivalent on ice for 30 minutes to preserve protein-protein interactions

• Pre-clearing: Pre-clear lysates with protein A/G-Sepharose beads at 4°C for 1 hour to reduce non-specific binding

• Immunoprecipitation:

  • Use 4 µg/ml of CREBBP antibody

  • Incubate overnight at 4°C with gentle rotation

  • Use appropriate IgG control (same host species) at equivalent concentration

• Bead Capture: Incubate protein-antibody complexes with protein A/G-Sepharose beads for 4 hours at 4°C

• Washing: Use stringent washing conditions (4-6 washes) with cold buffer

• Elution and Analysis: Elute under denaturing conditions and analyze by Western blotting

This approach has been validated for detecting interactions between CREBBP and transcription factors such as E2F3, which are critical in understanding CREBBP's role in transcriptional regulation .

What controls should be included when investigating CREBBP expression in patient samples?

When analyzing CREBBP expression in clinical specimens:

For immunohistochemistry, it is particularly important to optimize antigen retrieval methods, as CREBBP detection can be significantly affected by fixation and processing. Heat-mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes has been validated for formalin-fixed paraffin-embedded tissues .

How can I detect CREBBP loss in tumor samples when developing biomarkers for CDK4/6 inhibitor sensitivity?

To effectively detect CREBBP loss as a potential biomarker for CDK4/6 inhibitor sensitivity:

• Multi-modal approach: Combine genomic and protein analysis

  • Genomic: Assess for CREBBP mutations or deletions

  • Protein: Quantitative IHC for CREBBP protein expression

• IHC scoring system: Develop a standardized scoring system

  • Complete loss (0% positive cells)

  • Low expression (<20% positive cells)

  • Moderate expression (20-50% positive cells)

  • High expression (>50% positive cells)

• Validation cohort: Compare with known CREBBP wild-type and mutated samples

• Correlation analysis: Correlate CREBBP status with:

  • FOXM1 expression (elevated in CREBBP-deficient tumors)

  • Ki67 staining (increased in CREBBP-deficient tumors)

  • Patient outcomes data

This approach has been validated in triple-negative breast cancer models, where CREBBP loss was associated with upregulation of a FOXM1-driven proliferative program that rendered cells selectively sensitive to CDK4/6 inhibition .

How do CREBBP antibody staining patterns differ between normal B cells and lymphoma samples?

CREBBP expression patterns in normal versus lymphoma B cells show distinct characteristics:

When analyzing lymphoma samples, it's crucial to compare CREBBP staining with other markers including BCL2 (often overexpressed in conjunction with CREBBP loss). The pattern of CREBBP loss (complete vs. reduced) may have prognostic significance, as complete loss appears to correlate with more aggressive disease features .

How can I distinguish between CREBBP deficiency phenotypes in tumor samples versus technical artifacts?

To differentiate true CREBBP deficiency from technical artifacts:

• Multi-antibody approach: Use antibodies targeting different CREBBP epitopes

  • N-terminal epitopes (amino acids 150-200)

  • Mid-region epitopes (amino acids 451-682)

  • C-terminal epitopes (amino acids 2240-2441)

• Orthogonal validation:

  • Combine IHC with RNA analysis (qPCR or RNA-seq)

  • Validate with functional assays (e.g., acetylation of known CREBBP targets)

• Sample processing controls:

  • Include known positive samples processed simultaneously

  • Implement step-wise fixation time controls

  • Use dual chromogenic/fluorescent labeling to confirm specificity

• Internal controls: Look for non-tumor cells (stromal, inflammatory) within the same section that should maintain CREBBP expression as internal positive controls

These approaches help distinguish between true biological loss of CREBBP and technical artifacts that can occur with high molecular weight proteins like CREBBP (265 kDa) .

How can CREBBP antibodies be used to study its role in acetylation and lactylation of target proteins?

CREBBP functions as both a protein acetyltransferase and a protein lactyltransferase. To study these activities:

• Detection of modified targets:

  • Co-immunoprecipitate CREBBP with potential target proteins

  • Probe with pan-acetyl-lysine or pan-lactyl-lysine antibodies

  • Validate with site-specific acetylation/lactylation antibodies for known targets

• Functional studies:

  • Compare acetylation/lactylation patterns in CREBBP-proficient vs. deficient cells

  • Use CREBBP antibodies to confirm knockdown/knockout efficiency

  • Perform ChIP-seq with CREBBP antibodies to identify genomic binding sites

• Specifically for lactylation studies:

  • Focus on MRE11 lactylation in response to DNA damage

  • Monitor homologous recombination efficiency in relation to CREBBP expression

This approach has revealed that CREBBP can catalyze lactylation of MRE11 in response to DNA damage, promoting DNA double-strand break repair via homologous recombination .

What experimental approach should I use to investigate CREBBP's role in transcriptional regulation of cell cycle in cancer models?

To investigate CREBBP's role in cell cycle regulation:

• Gene silencing validation:

  • Use multiple shRNA/siRNA sequences targeting CREBBP

  • Confirm knockdown efficiency by Western blot using validated CREBBP antibodies

  • Compare protein and mRNA reduction levels

• Functional assays:

  • 3D spheroid growth assays to model in vivo conditions

  • Ki67 immunostaining to measure proliferation

  • Flow cytometry for cell cycle phase distribution

• Downstream target analysis:

  • Focus on E2F3 and FOXM1 as key CREBBP-regulated transcription factors

  • Examine expression of cell cycle genes (CASP8AP2)

  • Perform ChIP-seq to identify direct CREBBP binding sites at enhancers

• In vivo validation:

  • Generate Crebbp-deficient/BCL2-transgenic mouse models

  • Monitor for lymphoma development

  • Validate findings with patient-derived xenograft models

This comprehensive approach has revealed that CREBBP loss promotes cell cycle progression and proliferation in leukemia cells and enhances spheroid growth in breast cancer models, confirming its tumor-suppressive function in multiple cancer types .

How can I resolve discrepancies between CREBBP protein and mRNA expression data in experimental models?

When facing discrepancies between CREBBP protein and mRNA levels:

• Technical verification:

  • Confirm antibody specificity with positive and negative controls

  • Verify primer specificity for qPCR with melt curve analysis

  • Check for potential cross-reactivity with EP300 (p300), which shares significant homology

• Biological explanations:

  • Post-transcriptional regulation: Assess miRNA expression targeting CREBBP

  • Post-translational modifications: Examine ubiquitination status

  • Protein stability: Perform cycloheximide chase experiments to determine protein half-life

• Experimental design considerations:

  • Temporal dynamics: Different time points for mRNA vs. protein analysis

  • Cellular compartmentalization: Nuclear vs. cytoplasmic fraction analysis

  • Protein complexes: Native vs. denaturing conditions may affect antibody recognition

• Validation approach:

  • Use multiple antibodies recognizing different epitopes

  • Perform absolute quantification of transcript copy numbers

  • Implement mass spectrometry-based protein quantification

These approaches can help determine whether discrepancies represent biological phenomena or technical artifacts .

What are the critical parameters for optimizing chromatin immunoprecipitation (ChIP) using CREBBP antibodies?

For successful ChIP experiments with CREBBP antibodies:

When analyzing CREBBP binding to enhancer and super-enhancer regions, focus on genes involved in B-cell receptor signaling, CD40 receptor signaling, and transcriptional control of germinal center and plasma cell development, as these have been identified as key CREBBP-regulated networks .

How do I interpret contradictory findings regarding CREBBP's role as tumor suppressor versus oncogene across different cancer types?

When confronting seemingly contradictory roles of CREBBP across cancer types:

• Tissue context matters:

  • In B-cell lymphomas: CREBBP functions as a haploinsufficient tumor suppressor

  • In triple-negative breast cancer: CREBBP loss specifically promotes growth in 3D conditions

• Molecular context:

  • Examine co-occurring genetic alterations (e.g., BCL2 overexpression in lymphomas)

  • Assess FOXM1 pathway activation status

  • Evaluate p53 pathway integrity

• Functional readouts:

  • Growth conditions: 2D vs. 3D culture systems show different dependencies

  • Differentiation status: Effects on terminal differentiation programs

  • Cell cycle regulation: Impact on specific phase transitions

• Translational implications:

  • Different therapeutic vulnerabilities emerge:

    • CDK4/6 inhibitor sensitivity in CREBBP-deficient breast and lung cancers

    • HDAC inhibitor responses in CREBBP-mutant lymphomas

This nuanced approach recognizes that CREBBP functions in a highly context-dependent manner, with its tumor-suppressive or tumor-promoting effects depending on tissue type, molecular context, and microenvironmental conditions .

How should I reconcile different subcellular localization patterns observed with various CREBBP antibodies?

When observing different subcellular localization patterns:

• Epitope-specific considerations:

  • N-terminal antibodies may detect different CREBBP isoforms or fragments

  • C-terminal antibodies might be affected by post-translational modifications

  • Middle region antibodies could be influenced by protein-protein interactions

• Methodological factors:

  • Fixation methods: Paraformaldehyde vs. methanol have distinct effects

  • Permeabilization conditions: Triton X-100 concentration affects nuclear membrane permeability

  • Antigen retrieval: Different buffers (citrate pH 6.0 vs. TE pH 9.0) reveal different epitopes

• Biological explanations:

  • Cell cycle-dependent localization

  • Stimulus-responsive shuttling (e.g., DNA damage response)

  • Cell type-specific patterns

• Validation approaches:

  • Use tagged CREBBP constructs to confirm antibody findings

  • Perform subcellular fractionation followed by Western blotting

  • Implement super-resolution microscopy to resolve fine localization patterns