CEBPB Antibody

What is CEBPB and why is it important in research?

CEBPB (CCAAT/enhancer binding protein beta) is a critical transcription factor involved in regulating gene expression related to cell growth, differentiation, and immune responses. It plays a crucial role in modulating inflammatory processes and has been implicated in various physiological and pathological conditions . As a transcriptional activator, CEBPB regulates genes essential for cellular function, making it a significant target for researchers studying cell cycle regulation, inflammation, and cancer development .

What should researchers consider when selecting a CEBPB antibody?

When selecting a CEBPB antibody, researchers should consider several key factors: specificity, host species, clonality, and validated applications. The antibody's reactivity with target species (human, mouse, rat) must match the experimental model . For instance, antibody 23431-1-AP shows reactivity with human and mouse samples, while CAB0711 reacts with human, mouse, and rat samples . Additionally, the specific epitope region recognized by the antibody (such as AA 1-200 or AA 171-216) may impact detection efficiency depending on the protein conformation in your experimental conditions .

What are the typical molecular weights observed for CEBPB protein in Western blot applications?

While the calculated molecular weight of CEBPB is approximately 36 kDa (345 amino acids), researchers typically observe bands at 42-48 kDa in Western blot applications . This discrepancy between calculated and observed molecular weights is likely due to post-translational modifications or the presence of different isoforms. For example, in product 23431-1-AP, bands are commonly observed at 42 kDa and 46 kDa . Similar observations are noted with other antibodies, such as ABIN3043809, which detects CEBPB at approximately 48 kDa .

What are the recommended dilutions for different applications of CEBPB antibodies?

Different applications require specific antibody dilutions for optimal results. Based on validated data, the following dilution ranges are recommended:

It is important to note that these dilutions may need to be optimized based on your specific experimental conditions, sample type, and detection method . Always perform preliminary titration experiments to determine the optimal concentration for your system.

How should researchers optimize antigen retrieval for CEBPB immunohistochemistry?

For optimal antigen retrieval in CEBPB immunohistochemistry, using TE buffer at pH 9.0 is recommended as the primary method . If this does not yield satisfactory results, an alternative approach is to use citrate buffer at pH 6.0 . The efficiency of antigen retrieval significantly impacts staining intensity and specificity. Research indicates that CEBPB antibodies have been successfully used for detecting the protein in human colon cancer tissue using these retrieval methods . Temperature and duration of antigen retrieval should be optimized based on tissue type and fixation conditions to balance between adequate epitope exposure and tissue preservation.

What positive controls are recommended for validating CEBPB antibody performance?

To validate CEBPB antibody performance, several well-characterized cell lines have been documented as reliable positive controls:

These cell lines consistently express detectable levels of CEBPB protein and serve as appropriate benchmarks for antibody validation . For knockout/knockdown validation experiments, published literature includes at least 4 studies utilizing CEBPB knockdown approaches that can serve as reference methodologies .

How can researchers effectively use CEBPB antibodies in chromatin immunoprecipitation (ChIP) experiments?

CEBPB antibodies have been successfully employed in ChIP applications as documented in at least 8 published studies . For effective ChIP experiments, researchers should consider the following methodology:

• Cross-link protein-DNA complexes with 1% formaldehyde for 10 minutes at room temperature.

• Quench with 125 mM glycine for 5 minutes.

• Lyse cells and sonicate chromatin to fragments of 200-500 bp.

• Use 2-5 μg of CEBPB antibody per immunoprecipitation reaction.

• Include appropriate negative controls (IgG) and positive controls (input chromatin).

The high specificity of CEBPB antibodies for their target makes them valuable tools for identifying CEBPB binding sites across the genome . When designing ChIP experiments, consider the specific epitope recognized by the antibody to ensure it doesn't interfere with DNA binding domains.

What approaches should be used when investigating contradictory CEBPB function findings across different experimental systems?

When faced with contradictory findings regarding CEBPB function, researchers should implement a systematic approach to reconcile discrepancies:

• Compare experimental designs thoroughly, including cell types, expression levels, and temporal aspects of CEBPB activation.

• Utilize multiple antibodies recognizing different epitopes to verify results.

• Employ both in vitro and in vivo systems to validate findings.

• Consider generating genetic models (e.g., specific deletion variants) to test hypotheses.

As demonstrated in the literature, contradictory findings regarding CEBPB function may reflect differences in experimental design rather than actual biological discrepancies . For example, researchers investigating the proline-histidine-rich CDK2/CDK4 interaction region of C/EBPα found that in vitro results differed from in vivo knock-in mouse models, necessitating comprehensive validation across multiple systems .

How can researchers effectively use CEBPB antibodies in co-immunoprecipitation studies to identify interaction partners?

For effective co-immunoprecipitation (Co-IP) studies using CEBPB antibodies, the following methodology is recommended:

• Prepare cell lysates under non-denaturing conditions to preserve protein-protein interactions.

• Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Incubate cleared lysates with 2-5 μg of CEBPB antibody overnight at 4°C.

• Capture antibody-protein complexes with protein A/G beads for 2-4 hours.

• Wash extensively with buffers of decreasing stringency.

• Elute and analyze by Western blot using antibodies against suspected interaction partners.

CEBPB antibodies have been successfully used in at least 3 published Co-IP studies . When selecting antibodies for Co-IP, consider using those validated specifically for this application, as not all antibodies that work well in Western blot will perform adequately in Co-IP due to differences in epitope accessibility.

Why might Western blots with CEBPB antibodies show multiple bands, and how should these be interpreted?

Western blots using CEBPB antibodies frequently show multiple bands due to:

• Alternative translation initiation sites producing LAP* (38 kDa), LAP (35 kDa), and LIP (20 kDa) isoforms

• Post-translational modifications, particularly phosphorylation

• Proteolytic processing during sample preparation

The observed molecular weights typically range from 42-48 kDa compared to the calculated 36 kDa weight . To properly interpret these multiple bands:

• Use positive controls with known CEBPB expression patterns.

• Include phosphatase treatment controls to identify bands resulting from phosphorylation.

• Consider using isoform-specific antibodies when available.

• Compare band patterns with knockout/knockdown samples to confirm specificity.

The presence of specific isoforms may have biological significance, as different CEBPB isoforms can have distinct or even opposing functions in transcriptional regulation.

What are the optimal storage conditions for maintaining CEBPB antibody activity over time?

To maintain CEBPB antibody activity and prevent degradation, researchers should follow these storage guidelines:

• Store antibodies at -20°C for long-term preservation.

• Prepare small aliquots to avoid repeated freeze-thaw cycles.

• Most commercially available CEBPB antibodies are supplied in stabilizing buffers containing PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) .

How can specificity of CEBPB antibody staining be validated in immunohistochemistry applications?

To validate the specificity of CEBPB antibody staining in immunohistochemistry, researchers should implement a comprehensive approach:

• Include appropriate positive controls (tissues with known CEBPB expression).

• Use multiple antibodies targeting different epitopes of CEBPB.

• Implement peptide competition assays where pre-incubation of the antibody with the immunizing peptide should abolish specific staining.

• When possible, include tissues from knockout models or use siRNA-treated xenografts.

• Compare staining patterns with published literature and public databases.

For CEBPB immunohistochemistry, human colon cancer tissue has been validated as an appropriate positive control . The recommended antigen retrieval method using TE buffer (pH 9.0) or alternatively citrate buffer (pH 6.0) significantly impacts staining specificity . Additional validation may include correlation of staining intensity with quantitative PCR or Western blot analysis from the same tissue samples.

How are CEBPB antibodies being utilized in studies of cellular growth repression mechanisms?

CEBPB antibodies are increasingly important tools in investigating growth repression mechanisms, particularly in understanding the function of CEBPB's homolog C/EBPα. Research methodologies include:

• Generating C/EBPα-ER fusion proteins for tamoxifen-inducible studies

• Using BrdU pulse-labeling (45 minutes) to assess S-phase entry

• Employing FACS analysis to evaluate cell cycle distribution

• Creating targeted deletion variants to identify functional domains

Recent studies have challenged previous findings regarding the importance of specific domains (such as the proline-histidine-rich region) in growth repression, highlighting the need for comprehensive in vitro and in vivo validation approaches . When using antibodies to study CEBPB/C/EBPα-mediated growth repression, researchers typically maintain cells at subconfluency for 2-3 weeks with regular trypsinization and replating to accurately assess long-term effects on proliferation .

What is the role of CEBPB in immune responses and inflammation, and how can antibodies help elucidate these mechanisms?

CEBPB plays a crucial role in immune response regulation and inflammation through:

• Transcriptional control of cytokine production

• Regulation of macrophage activation and polarization

• Modulation of inflammatory signaling pathways

CEBPB antibodies enable researchers to investigate these mechanisms through:

• ChIP studies to identify inflammatory gene targets directly regulated by CEBPB

• Immunofluorescence to track CEBPB nuclear translocation during immune cell activation

• Co-IP experiments to identify interaction partners in inflammatory signaling cascades

• Tissue staining to assess CEBPB expression in models of inflammatory diseases

Research indicates that CEBPB functions as an important transcriptional activator in the regulation of genes involved in immune and inflammatory responses . By using CEBPB antibodies in these applications, researchers can map the complex regulatory networks controlled by this transcription factor in various immune contexts.