CEBPA Antibody

What is CEBPA and what are its primary biological functions?

CEBPA is a transcription factor that coordinates proliferation arrest and the differentiation of various cell types including myeloid progenitors, adipocytes, hepatocytes, and cells of the lung and placenta. It binds directly to the consensus DNA sequence 5'-T[TG]NNGNAA[TG]-3' and acts as an activator on distinct target genes . CEBPA plays essential roles in several biological processes:

• During early embryogenesis, it has essential and redundant functions with CEBPB

• It is essential for the transition from common myeloid progenitors (CMP) to granulocyte/monocyte progenitors (GMP)

• It is critical for proper development of the liver and lung

• It is necessary for terminal adipocyte differentiation and maintenance of systemic energy homeostasis

• In the liver, it regulates gluconeogenesis and lipogenesis through different mechanisms

CEBPA interacts with various transcription factors including FOXO1, SREBF1, and E2F1 to regulate different processes in a tissue-specific manner .

How should I select the appropriate CEBPA antibody for my specific application?

Selection of the appropriate CEBPA antibody should be based on several key factors:

• Experimental application: Different antibodies perform optimally in specific applications. For example:

  • For Western blotting: Most antibodies show good reactivity, with recommended dilutions typically between 1:500-1:1000

  • For ChIP applications: Specially validated antibodies like 18311-1-AP have been cited in multiple publications

  • For immunofluorescence: Antibodies like #2295 have recommended dilutions of 1:50

• Target species: Verify reactivity with your experimental model. Most CEBPA antibodies react with human, mouse, and rat samples, but validation in other species may be limited .

• Specific CEBPA domain or isoform: Consider whether you need to detect:

  • Total CEBPA protein (use antibodies targeting conserved regions)

  • Specific phosphorylated forms (use phospho-specific antibodies for pSer21 or pThr226)

  • N-terminal or C-terminal regions (particularly important when studying mutations or truncated forms)

• Clonality preference: Monoclonal antibodies offer higher specificity, while polyclonal antibodies may provide stronger signals by recognizing multiple epitopes .

• Validation data: Review available validation data including knockout/knockdown controls, which are critical for confirming specificity .

For most comprehensive studies of CEBPA, researchers should consider having both a general anti-CEBPA antibody and one or more phospho-specific antibodies to capture the complete regulatory picture.

What controls should be included when validating a CEBPA antibody for research?

Proper validation of CEBPA antibodies requires several essential controls:

• Positive tissue/cell controls:

  • Human liver tissue or L02 cells express detectable levels of endogenous CEBPA

  • Adipocyte, myeloid cell lines, and hepatocytes are suitable positive controls

• Negative controls:

  • CEBPA knockout or knockdown samples are ideal negative controls

  • In ChIP experiments, researchers have confirmed antibody specificity by performing ChIP in mice deficient for Cebpa or Cebpb

• Peptide competition assays:

  • Including epitope blocking peptides to confirm binding specificity

  • This technique has been used successfully in ChIP validation

• Molecular weight verification:

  • CEBPA typically appears at 42 kDa (full-length p42 isoform) and 28-30 kDa (p30 isoform)

  • Some studies observe CEBPA at 40-45 kDa

• Cross-reactivity testing:

  • Test against related family members (CEBPB, CEBPD, etc.) to ensure specificity

For ChIP applications specifically, de novo motif searches on ChIP-seq data and conservation score analysis centering on CEBP motifs have been used to further confirm antibody quality .

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

For optimal Western blotting using CEBPA antibodies, follow these methodological guidelines:

• Sample preparation:

  • Use whole-cell lysates (50 µg of extracted proteins is typically sufficient)

  • Include protease and phosphatase inhibitors to prevent degradation and preserve phosphorylation states

• Antibody dilution:

  • Most CEBPA antibodies work efficiently at dilutions of 1:500-1:1000

  • Optimize dilution for each new lot of antibody

• Detection considerations:

  • Expected molecular weights: 42 kDa (p42 full-length isoform) and 28 kDa (p30 truncated isoform)

  • Some antibodies may detect CEBPA at 40-45 kDa

  • N-terminal mutations can result in expression of only the p30 isoform, while C-terminal mutations affect DNA binding capacity

• Membrane blocking:

  • BSA-based blocking solutions are generally preferred over milk for phospho-specific CEBPA antibodies

• Validation strategies:

  • Include positive controls like liver tissue or L02 cells

  • For complete validation, include CEBPA-knockout samples when available

When studying mutant CEBPA proteins, be aware that N-terminal frame-shift mutations and C-terminal in-frame mutations can affect protein detection patterns, which is crucial for research on conditions like AML with CEBPA mutations .

How should CEBPA antibodies be used in chromatin immunoprecipitation (ChIP) experiments?

CEBPA antibodies have been extensively used in ChIP experiments to study transcription factor binding dynamics. Here's a methodological approach based on published protocols:

• Antibody selection:

  • Choose antibodies specifically validated for ChIP applications, such as 18311-1-AP

  • Confirm antibody specificity using knockout controls or epitope blocking peptides

• Experimental protocol:

  • For liver tissue ChIP, pooling samples from multiple mice (e.g., five per time point) helps minimize experimental variation

  • Equal amounts of precipitated DNA should be pooled and sequenced

  • Include mock immunoprecipitation controls for peak-finding algorithms

• Data analysis approach:

  • Map sequences to the appropriate genome (e.g., mm9 for mouse studies)

  • Normalize to total mapped read count for each antibody

  • Use peak-finder algorithms (e.g., Useq) with appropriate controls

• Validation of ChIP quality:

  • Perform de novo motif searches on data sets

  • Conduct conservation score analysis centering on CEBP motifs

  • Validate consistency with independent ChIPs at different CEBP-bound genomic locations

In temporal mapping studies, CEBPA binding has been shown to exhibit distinct patterns associated with specific sets of regulated genes, making time-course ChIP experiments particularly valuable for understanding CEBPA's dynamic regulatory roles .

How are CEBPA antibodies used to characterize mutations in acute myeloid leukemia (AML)?

CEBPA mutations in AML are associated with favorable prognosis and are divided into N-terminal and C-terminal mutations. Antibodies play a crucial role in characterizing these mutations:

• Mutation classification and detection:

  • N-terminal mutations typically lead to production of the p30 isoform only

  • C-terminal mutations affect DNA binding capacity

  • Western blotting with antibodies recognizing different CEBPA domains can help identify mutation patterns

• Functional characterization:

  • Research has shown that both N- and C-terminal CEBPA mutant peptides inhibit wild-type CEBPA protein in a dominant-negative manner, reducing its activation potential by approximately 70%

  • The combination of both mutant constructs (double mutations) fails to activate target genes at all

• Prognostic implications:

  • Patients with double CEBPA mutations show completely abolished CEBPA activity

  • Patients with single CEBPA mutations retain some degree of CEBPA activity

  • This distinction has important clinical implications, as heterogeneity within AML with CEBPA mutations affects patient outcomes

• Experimental validation:

  • Reporter gene assays using constructs with CEBPA binding sites can be used to assess the functional impact of mutations

  • H1299 cells transfected with expression plasmids encoding CEBPA wild-type or mutant forms have been used to study the activation potential of these CEBPA peptides on target promoter sequences

This research demonstrates how antibodies enable both the detection and functional characterization of CEBPA mutations, contributing to better understanding of AML pathogenesis and prognosis.

What role does CEBPA play in endometrial cancer and how can antibodies help study this connection?

Recent research has identified important roles for CEBPA in uterine corpus endometrial carcinoma (UCEC), where antibody-based techniques provide critical insights:

These findings suggest CEBPA may be a potential therapeutic target in UCEC, highlighting how antibody-based research expands our understanding of CEBPA's roles beyond traditional contexts into cancer biology.

What are common issues encountered when using CEBPA antibodies and how can they be resolved?

Researchers commonly encounter several challenges when working with CEBPA antibodies:

• Multiple band detection in Western blotting:

  • Issue: Detecting multiple bands beyond the expected 42 kDa (p42) and 28-30 kDa (p30) isoforms

  • Solution: Verify antibody specificity using knockout/knockdown controls; test blocking peptides; optimize protein extraction protocols to minimize degradation; include phosphatase treatment to determine if bands represent phosphorylated forms

• Low signal intensity:

  • Issue: Weak detection of CEBPA despite appropriate sample selection

  • Solution: Optimize antibody concentration; increase protein loading; extend exposure time; use enhanced chemiluminescence detection systems; consider alternative antibodies targeting different epitopes

• Cross-reactivity with other C/EBP family members:

  • Issue: Antibodies detecting related family members (CEBPB, CEBPD, etc.)

  • Solution: Select antibodies raised against unique regions of CEBPA; validate specificity using recombinant proteins of multiple C/EBP family members; include appropriate controls expressing only specific C/EBP proteins

• ChIP-seq background noise:

  • Issue: High background in ChIP experiments affecting peak identification

  • Solution: Include mock immunoprecipitation controls; perform de novo motif searches on datasets; conduct conservation score analysis centering on CEBP motifs; validate with independent ChIPs at different CEBP-bound genomic locations

• Inconsistent results between applications:

  • Issue: Antibody works in Western blot but not in immunohistochemistry or ChIP

  • Solution: Select application-specific validated antibodies; adjust fixation protocols for IHC; optimize chromatin shearing for ChIP; consider epitope accessibility in different applications

Each troubleshooting approach should be methodically tested and documented to establish reliable protocols for CEBPA detection in specific experimental contexts.

How can researchers optimize immunohistochemistry and immunofluorescence protocols for CEBPA detection?

Optimizing immunohistochemistry (IHC) and immunofluorescence (IF) protocols for CEBPA detection requires attention to several methodological details:

• Tissue preparation and fixation:

  • Formalin-fixed paraffin-embedded (FFPE) tissues typically require antigen retrieval

  • For CEBPA, heat-induced epitope retrieval in citrate buffer (pH 6.0) often yields optimal results

  • Fresh frozen sections may provide better epitope preservation for certain antibodies

• Antibody selection and dilution:

  • For IF applications, antibodies like #2295 have been validated at 1:50 dilution

  • For IHC, multiple antibodies have been validated, with application-specific recommended dilutions

  • Always perform a dilution series to determine optimal concentration for your specific tissue and fixation method

• Signal amplification strategies:

  • Consider tyramide signal amplification for low abundance transcription factors like CEBPA

  • Polymer-based detection systems often provide better signal-to-noise ratio than standard ABC methods

• Controls for validation:

  • Positive controls: Liver tissue shows strong endogenous CEBPA expression

  • Negative controls: Include CEBPA-deficient tissues or primary antibody omission

  • Peptide competition: Pre-incubating antibody with immunizing peptide should eliminate specific staining

• Nuclear staining optimization:

  • As CEBPA is a transcription factor, nuclear localization should be evident

  • Counterstain with DAPI or similar nuclear stains to confirm nuclear localization

  • Consider membrane permeabilization optimization to ensure nuclear epitope accessibility

• Dual staining approaches:

  • When studying cell-type specific expression, combine CEBPA detection with lineage markers

  • Use sequential staining protocols to avoid cross-reactivity between detection systems

These optimizations are particularly important when studying CEBPA in disease contexts such as AML with CEBPA mutations or UCEC where CEBPA upregulation has prognostic significance .

How might emerging technologies enhance the application of CEBPA antibodies in research?

Several emerging technologies have potential to significantly advance CEBPA antibody applications in research:

• Single-cell technologies:

  • Integration of CEBPA antibodies with single-cell Western blotting can reveal cell-to-cell variability in CEBPA expression and isoform usage

  • Single-cell CUT&Tag or CUT&RUN approaches could map CEBPA binding sites at single-cell resolution, revealing heterogeneity in transcription factor activity

• Proximity labeling approaches:

  • BioID or APEX2 fusions with CEBPA could identify context-specific protein interaction partners

  • This approach could help elucidate how CEBPA functions differently across various tissues and disease states

• Live-cell imaging of CEBPA dynamics:

  • Development of nanobodies or intrabodies against CEBPA could enable live visualization of CEBPA activity

  • Combining with lattice light-sheet microscopy could reveal dynamic binding events in living cells

• Spatial transcriptomics integration:

  • Combining CEBPA immunodetection with spatial transcriptomics could map relationships between CEBPA localization and target gene expression within tissue architecture

  • This would be particularly valuable in heterogeneous tissues like liver and in cancer contexts

• High-throughput antibody validation platforms:

  • CRISPR-based knockout cell arrays for systematic validation of antibody specificity

  • Automated immunoprecipitation-mass spectrometry workflows to identify antibody binding partners

These technological advances could substantially enhance our understanding of how CEBPA functions in different cellular contexts and disease states, potentially revealing new therapeutic strategies for conditions like AML with CEBPA mutations or UCEC where CEBPA upregulation affects prognosis .

What are emerging research questions regarding CEBPA function that could benefit from improved antibody-based approaches?

Several key research questions about CEBPA function remain to be fully addressed through improved antibody-based approaches:

• Temporal dynamics of CEBPA binding and activity:

  • How does CEBPA binding change during differentiation or disease progression?

  • Building on temporal mapping studies , high-resolution time-course analyses using ChIP-seq could reveal dynamic regulatory networks

  • Antibodies recognizing specific post-translational modifications could track how CEBPA activity is regulated over time

• CEBPA isoform-specific functions:

  • How do the p42 and p30 isoforms differ functionally beyond what's currently understood?

  • Isoform-specific antibodies could help distinguish unique binding partners and genomic targets

  • This is particularly relevant for understanding leukemogenesis in AML with CEBPA mutations

• Tissue-specific CEBPA interactome:

  • How does the CEBPA protein interaction network differ across tissues?

  • Antibody-based co-immunoprecipitation coupled with mass spectrometry in different tissue contexts could reveal tissue-specific regulatory mechanisms

  • This could explain how CEBPA regulates distinct processes in liver versus adipose tissue versus myeloid cells

• CEBPA in immune modulation:

  • Building on findings in UCEC , how does CEBPA influence immune cell infiltration and function in different cancers?

  • Multiplexed immunohistochemistry using CEBPA antibodies alongside immune cell markers could map spatial relationships

  • This could reveal new immunotherapeutic approaches for CEBPA-dysregulated cancers

• CEBPA in therapeutic response prediction:

  • Can CEBPA expression or phosphorylation status predict response to specific therapies?

  • Antibody-based tissue microarray studies correlating CEBPA levels with treatment outcomes could identify predictive biomarkers

  • This approach could help stratify patients for personalized treatment approaches