CREB1 Monoclonal Antibody

What is CREB1 and why is it important in cellular biology?

CREB1 is a phosphorylation-dependent transcription factor that binds to the cAMP response element (CRE) in response to specific cellular stimuli. It contains a basic leucine zipper (bZIP) DNA-binding domain, a dimerization domain, and a kinase-inducible domain with nine serine residues that can be phosphorylated by multiple kinases, including CaMK II and IV, PKA, PKC, MSK, RSK, AKT, and MK2 . CREB1 is crucial for maintaining cellular homeostasis in both physiological and pathological conditions . Recent research has demonstrated that CREB1 plays a significant role in immunogenicity and may be a mechanistic driver of reduced HIV-1 acquisition following vaccination . Due to its involvement in numerous signaling pathways and cellular processes, CREB1 has become a vital research target across multiple fields including neuroscience, immunology, and cancer biology.

In which tissues and cell types is CREB1 typically expressed?

CREB1 shows widespread expression across multiple tissues and cell types. According to literature data and antibody validation studies, CREB1 expression has been confirmed in:

Researchers should note that CREB1 is primarily localized in the nucleus, which is consistent with its function as a transcription factor . When designing experiments to detect CREB1, subcellular localization is an important consideration, particularly when using imaging techniques such as immunofluorescence or immunohistochemistry.

What are the key differences between various CREB1 antibody types?

CREB1 antibodies can be categorized based on several characteristics that impact their research applications:

When selecting a CREB1 antibody, researchers should carefully consider the experimental question at hand. For monitoring phosphorylation-dependent activation, a phospho-specific antibody like anti-phospho-CREB (S133) is appropriate . For general expression studies, a pan-CREB1 antibody that detects total CREB1 regardless of modification status would be more suitable .

What are the validated applications for CREB1 monoclonal antibodies?

CREB1 monoclonal antibodies have been validated for numerous experimental applications. Below is a comprehensive table of validated applications based on the provided search results:

For optimal results in Western blotting, researchers should expect to observe CREB1 at approximately 43-46 kDa, which is slightly higher than the calculated molecular weight of 35 kDa due to post-translational modifications . When performing immunofluorescence or immunohistochemistry, nuclear staining pattern is expected due to CREB1's function as a transcription factor .

How should I optimize Western blot protocols for CREB1 detection?

Optimizing Western blot protocols for CREB1 detection requires attention to several key factors:

• Sample Preparation:

  • Include phosphatase inhibitors in lysis buffer if detecting phosphorylated CREB1

  • Nuclear extraction protocols may yield cleaner results for CREB1 detection

  • Ensure adequate denaturation of samples (95°C for 5 minutes in loading buffer)

• Gel Separation and Transfer:

  • Use 10-12% polyacrylamide gels for optimal separation around 43-46 kDa (observed molecular weight)

  • Transfer conditions: 100V for 60-90 minutes or 30V overnight for complete transfer

• Antibody Incubation:

  • Primary antibody dilution: Typically 1:1000 to 1:2000 in 5% BSA or non-fat milk

  • Incubation time: Overnight at 4°C yields optimal signal-to-noise ratio

  • Secondary antibody: HRP-conjugated anti-mouse IgG for monoclonal antibodies like clone A18233D

• Troubleshooting Common Issues:

  • Multiple bands: May indicate protein degradation or cross-reactivity

  • Weak signal: Increase antibody concentration or protein loading

  • High background: Increase blocking time or washing steps

When validating a new CREB1 antibody, researchers should include positive control samples from tissues known to express CREB1, such as brain tissue or neuronal cell lines. The expected molecular weight for CREB1 is approximately 43-46 kDa, which is higher than the calculated 35 kDa due to post-translational modifications .

What considerations are important for immunohistochemistry with CREB1 antibodies?

For optimal immunohistochemistry (IHC) results with CREB1 antibodies, researchers should consider:

• Fixation and Antigen Retrieval:

  • Formalin-fixed paraffin-embedded (FFPE) tissues typically require heat-induced epitope retrieval

  • Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) are commonly effective for CREB1 epitope retrieval

  • For phospho-specific antibodies (e.g., p-CREB S133), antigen retrieval conditions may need further optimization

• Antibody Selection and Controls:

  • Validate antibody specificity using tissues with known CREB1 expression patterns

  • Include both positive controls (e.g., testis or cervix carcinoma) and negative controls

  • For phospho-CREB detection, consider using paired specimens with and without stimulation of relevant signaling pathways

• Staining Pattern Interpretation:

  • CREB1 shows predominantly nuclear localization

  • In testis and cervix carcinoma, positive nuclear staining has been consistently observed

  • When using phospho-specific antibodies, staining intensity may vary with activation state

• Quantification Approaches:

  • H-score (combining intensity and percentage of positive cells)

  • Nuclear labeling index (percentage of positive nuclei)

  • Digital image analysis for more objective quantification

Researchers investigating CREB1 expression in rat testis have reported positive staining , consistent with literature evidence from multiple studies (PubMed ID: 15579595). When working with new tissue types, literature validation and appropriate controls are essential for accurate interpretation of results.

How can I distinguish between CREB1 and closely related family members?

Distinguishing between CREB1 and its closely related family members (CREM and ATF1) requires careful antibody selection and experimental design:

• Epitope Selection Considerations:

  • CREB1, CREM, and ATF1 share high sequence homology in their bZIP and kinase-inducible domains

  • Monoclonal antibodies targeting unique regions outside these conserved domains offer higher specificity

  • Clone A18233D targets specific epitopes in CREB1 that minimize cross-reactivity

• Validation Strategies:

  • Overexpression systems with tagged variants of each family member

  • Knockout or knockdown approaches to confirm specificity

  • Peptide competition assays with family-specific peptide sequences

• Technical Approaches to Resolve Ambiguity:

  • Western blot resolution based on subtle molecular weight differences (CREB1: 43-46 kDa)

  • Two-dimensional gel electrophoresis for isoform separation

  • Immunoprecipitation followed by mass spectrometry analysis

• Functional Assays for Distinction:

  • Family member-specific reporter constructs

  • ChIP-seq to identify binding site preferences

  • Co-immunoprecipitation with known specific interaction partners

Researchers should be aware that some commercially available antibodies may cross-react with multiple CREB family members. When absolute specificity is required, validation using knockout cell lines or tissues is highly recommended, along with careful examination of the antibody epitope information provided by manufacturers.

What is the significance of CREB1 phosphorylation at Serine 133 in different cellular contexts?

Phosphorylation of CREB1 at Serine 133 (p-CREB S133) is a critical regulatory event with context-dependent significance:

• Signaling Pathways Leading to S133 Phosphorylation:

  • cAMP-dependent protein kinase A (PKA)

  • Ca²⁺/calmodulin-dependent protein kinases (CaMKs)

  • Ribosomal S6 kinases (RSKs)

  • Mitogen/stress-activated kinases (MSKs)

  • Protein kinase C (PKC)

  • AKT and MAPKAP kinase 2 (MK2)

• Functional Consequences in Different Tissues:

  • Neural tissues: Memory formation and synaptic plasticity

  • Immune cells: Modulation of cytokine production and immune responses

  • HIV vaccination responses: Enhanced immunogenicity and reduced viral acquisition

  • Circadian rhythm regulation: Phosphorylation of both Ser-133 and Ser-142 in the suprachiasmatic nucleus (SCN)

• Regulatory Mechanisms Beyond Phosphorylation:

  • Interaction with CREB-binding protein (CBP) is enhanced by S133 phosphorylation

  • TORC coactivators can enhance transcription independently of S133 phosphorylation

  • Sumoylation at Lys-304 (enhanced under hypoxia) affects nuclear localization and stability

In the context of HIV vaccination, research has demonstrated that CREB1 activity and its target genes following ALVAC+Alum vaccination show significant association with reduced HIV-1 acquisition in the RV144 trial . The expression of CREB1 targets was significantly elevated in vaccinated participants who remained uninfected compared to those who became infected post-vaccination . This highlights the importance of considering the specific cellular and physiological context when studying CREB1 phosphorylation patterns.

How does CREB1 signaling influence immune responses in the context of vaccination?

Recent research has revealed significant insights into CREB1's role in immune responses, particularly in the context of HIV vaccination:

• Transcriptional Regulation of Immune Mediators:

  • CREB1 drives expression of cytokines and chemokines associated with protection from SIV challenge in Non-human Primates (NHPs)

  • Key CREB1-regulated immune mediators include Fractalkine (CX3CL1), GROα (CXCL1), MCP1, FLT3LG, and TGF-β1/3

  • These factors were identified as significant positive correlates of protective V1V2 antibody responses in Study 36

• Adjuvant-Dependent CREB1 Activation:

  • ALVAC+Alum vaccination regimen (used in RV144 trial) effectively induced CREB1 signaling

  • ALVAC+MF59 regimen (used in HVTN702 trial, which showed no protection) exhibited significantly reduced CREB1 target gene expression

  • cGAMP (STING agonist) modulates p-CREB1 activity, driving recruitment of CD4+ T cells and B cells to antigen presentation sites

• Correlation with Protection Metrics:

  • Kaplan-Meier analysis of RV144 trial participants revealed significantly reduced risk of HIV-1 acquisition in subjects with medium and high CREB1 z-scores

  • High CREB1 z-score participants maintained lower acquisition risk up to three years post-vaccination

  • In NHP studies, CREB1 gene expression positively correlated with rectal IgG against cV2 and other SIV-specific IgGs

• Chemokine-Mediated Migration Mechanisms:

  • CREB1-associated chemokines promote enhanced expression of chemotaxis and GPCR signaling pathways in DCs, CD4+ T cells, and B cells post-vaccination

  • Fractalkine (CX3CL1), an inducer of monocyte migration, showed positive correlation with the number of challenges needed to infect NHPs producing cV2-specific antibodies

  • Conversely, Eotaxin-3 (CCL26), a repressor of monocyte migration, was a negative correlate of protection

This evidence suggests that adjuvants triggering CREB1 signaling may be critical for developing efficacious HIV-1 vaccines. Researchers investigating CREB1's role in vaccination responses should consider monitoring both transcriptional signatures and functional outcomes like chemokine production and cellular migration patterns.

What are common pitfalls when using CREB1 monoclonal antibodies and how can they be avoided?

Researchers frequently encounter several challenges when working with CREB1 monoclonal antibodies. Here are common pitfalls and their solutions:

• False Negative Results:

  • Pitfall: Inadequate sample preparation leading to epitope masking

  • Solution: Optimize fixation and antigen retrieval conditions; for phospho-specific antibodies, ensure samples are collected with phosphatase inhibitors and processed quickly

• Cross-reactivity Issues:

  • Pitfall: Antibody detecting related family members (ATF1, CREM)

  • Solution: Validate antibody specificity using knockout/knockdown controls; select monoclonal antibodies with verified specificity like clone A18233D

• Inconsistent Phosphorylation Detection:

  • Pitfall: Rapid dephosphorylation during sample handling

  • Solution: Maintain samples at 4°C; include phosphatase inhibitors in all buffers; consider using phospho-mimetic controls for validation

• Background Staining in Immunohistochemistry:

  • Pitfall: Non-specific binding, particularly in certain tissues

  • Solution: Optimize blocking conditions (5% BSA or 10% normal serum); include appropriate negative controls; consider using monoclonal antibodies for higher specificity

• Unexpected Staining Patterns:

  • Pitfall: Observing positive staining in tissues where CREB1 expression was not anticipated

  • Solution: Consult literature for comprehensive expression data ; as noted in customer Q&As, CREB1 expression has been confirmed in diverse tissues including cervix carcinoma, testis, eye, and liver

• Molecular Weight Discrepancies:

  • Pitfall: Observed molecular weight (43-46 kDa) differs from calculated weight (35 kDa)

  • Solution: Be aware that post-translational modifications affect migration; use positive control lysates with confirmed CREB1 expression

For optimal results, researchers should conduct preliminary validation experiments to determine ideal antibody concentrations, incubation conditions, and detection methods for their specific experimental system and antibody clone.

How can I design experiments to study CREB1 activation in response to specific stimuli?

Designing robust experiments to study CREB1 activation requires careful consideration of temporal dynamics, pathway specificity, and appropriate readouts:

• Stimulus Selection and Timing:

  • cAMP pathway activators: Forskolin (adenylyl cyclase activator), dibutyryl-cAMP, or GPCR agonists

  • Ca²⁺-dependent pathways: Ionomycin, KCl (for neuronal depolarization), or specific receptor agonists

  • Growth factor pathways: EGF, BDNF, or insulin for RSK/MSK-mediated activation

  • Time course considerations: Early phosphorylation (5-30 minutes) → nuclear translocation → target gene expression (1-4 hours)

• Phosphorylation Analysis Approaches:

  • Western blotting: Anti-phospho-CREB (S133) antibodies with total CREB normalization

  • Flow cytometry: For single-cell resolution of phosphorylation events

  • Immunofluorescence: To visualize subcellular localization changes

  • Phospho-protein arrays: For multiplexed analysis of CREB1 and related pathway components

• Transcriptional Activation Measurement:

  • RT-qPCR of known CREB1 target genes: BDNF, c-fos, PEPCK, somatostatin

  • CRE-luciferase reporter assays: Direct measurement of CREB-dependent transcription

  • ChIP assays: To assess CREB1 binding to specific promoters

  • RNA-seq with bioinformatic analysis: For genome-wide identification of CREB1-responsive genes

• Pathway Validation Strategies:

  • Pharmacological inhibitors: H89 (PKA), KN-93 (CaMK), U0126 (MEK/ERK pathway)

  • Genetic approaches: Dominant-negative CREB, CREB1 knockdown/knockout

  • Mutations at key residues: S133A (phosphorylation-deficient) or S133D (phosphomimetic)

• Sample Experimental Design for HIV Vaccine Research:

  • Based on findings from the RV144 trial, researchers could design experiments to:

  • Compare CREB1 activation between different adjuvant formulations (Alum vs. MF59)

  • Measure downstream cytokine/chemokine production, particularly Fractalkine (CX3CL1), GROα (CXCL1), and MCP1

  • Assess the impact on immune cell migration and chemotaxis using transwell assays

  • Correlate CREB1 activation with antibody responses and protection metrics in animal models

When designing these experiments, researchers should include appropriate positive controls (stimulus known to activate CREB1) and negative controls (pathway inhibitors or phosphorylation-deficient mutants) to validate the specificity of observed responses.

What are the best practices for quantifying CREB1 expression and activation in multicellular systems?

Quantifying CREB1 expression and activation in complex multicellular systems presents unique challenges that require specialized approaches:

• Tissue-Specific Expression Analysis:

  • Immunohistochemistry with digital quantification: Use algorithms that can distinguish nuclear staining intensity across different cell types

  • Laser capture microdissection: For isolating specific cell populations before protein/RNA extraction

  • Single-cell RNA sequencing: To determine cell-type-specific CREB1 expression patterns

  • Flow cytometry with cell type markers: For quantitative analysis of CREB1 levels in distinct cell populations

• Activation State Assessment in Heterogeneous Samples:

  • Phospho-flow cytometry: Combines surface markers with intracellular p-CREB (S133) staining

  • Multiplexed immunofluorescence: Co-localization of p-CREB with cell type-specific markers

  • CyTOF (mass cytometry): For high-dimensional analysis of signaling states in complex samples

  • Spatial transcriptomics: To map CREB1 target gene expression in tissue context

• Normalization Strategies for Comparative Studies:

  • Ratio of phospho-CREB to total CREB: Accounts for expression level differences

  • Cell type-specific housekeeping genes: When analyzing sorted populations

  • CREB1 gene dose normalization: Important when comparing samples with potential copy number variations

  • Standardized positive controls: Samples treated with forskolin or other known CREB1 activators

• Application to Vaccination Studies:

  • In HIV vaccine research, CREB1 activity has been quantified using:

  • CREB1 geneset z-scores calculated from transcriptomic data

  • Measurement of downstream cytokines/chemokines in plasma

  • Correlation of CREB1 activity with protection metrics (challenges to infection)

• Statistical Considerations for Complex Data:

  • Multiple testing correction: Essential when analyzing many cell types or conditions

  • Nested models for repeated measures: When following activation over time

  • Correlation with functional outcomes: As demonstrated in RV144 trial analysis where CREB1 z-scores correlated with protection

  • Machine learning approaches: For identifying patterns in high-dimensional CREB1 signaling data

When quantifying CREB1 in multicellular systems, researchers should consider cell type-specific baseline expression levels. For example, studies have documented CREB1 expression in diverse tissues including cervix carcinoma, testis, and liver , but expression levels and activation states may vary significantly among different cell types within these tissues.

How are CREB1 monoclonal antibodies being used in current immunotherapy research?

CREB1 monoclonal antibodies are increasingly valuable tools in immunotherapy research, with several emerging applications:

• HIV Vaccine Development:

  • CREB1 signaling has been identified as a mechanistic driver of immunogenicity in HIV vaccination

  • Monitoring CREB1 activation via phospho-specific antibodies helps evaluate adjuvant efficacy

  • CREB1-regulated cytokines and chemokines (e.g., Fractalkine/CX3CL1) correlate with protection in NHP models

  • High CREB1 activity signature maintained protection for up to three years in RV144 vaccine recipients

• Cancer Immunotherapy Biomarkers:

  • CREB1 activity in tumor-infiltrating lymphocytes may predict immunotherapy response

  • Phospho-CREB antibodies enable monitoring of T cell activation states

  • Chromosomal aberrations involving CREB1, such as the t(2;22)(q33;q12) translocation generating EWSR1/CREB1 fusion gene, have been found in angiomatoid fibrous histiocytoma patients

• Modulation of Immune Cell Migration:

  • CREB1-regulated chemokines influence immune cell trafficking to vaccination sites

  • Anti-CREB1 antibodies help track activation of migratory programs in dendritic cells and T cells

  • CREB1 target genes including chemokines are associated with enhanced expression of chemotaxis and GPCR signaling pathways in DCs, CD4+ T cells, and B cells post-vaccination

• Adjuvant Development and Screening:

  • CREB1 phosphorylation as a readout for adjuvant activity

  • Comparison of different adjuvant formulations based on CREB1 activation profiles

  • The differing effects of Alum versus MF59 on CREB1 signaling pathways may explain efficacy differences between vaccination regimens

Research has demonstrated that CREB1-driven genes are induced early post-immunization with ALVAC, with induction persisting over time (up to 3 days) across consecutive vaccinations . This highlights CREB1's central role in modulating HIV-1 vaccine responses and suggests potential applications in designing more effective vaccination strategies.

What novel methods are being developed to study CREB1 dynamics in living cells?

Cutting-edge techniques are advancing our ability to study CREB1 dynamics with unprecedented temporal and spatial resolution:

• Real-time Imaging Technologies:

  • FRET-based CREB1 biosensors: For visualizing phosphorylation dynamics in living cells

  • Split-luciferase complementation systems: To monitor CREB1-CBP interactions

  • Fluorescently-tagged CREB1: For tracking nuclear translocation kinetics

  • Optogenetic CREB1 activation systems: For spatiotemporal control of CREB1 function

• Proximity Labeling Approaches:

  • BioID or TurboID-CREB1 fusions: For identifying context-specific interaction partners

  • APEX2-based proximity labeling: To map the CREB1 protein neighborhood in different activation states

  • Split-BioID systems: To capture transient CREB1-coactivator interactions

• Advanced Genomic Technologies:

  • CUT&RUN or CUT&Tag: For high-resolution mapping of CREB1 binding sites

  • CRISPR activation/repression systems: To modulate CREB1 activity at specific target genes

  • Single-cell multi-omics: To correlate CREB1 binding, chromatin accessibility, and gene expression

• Computational Approaches:

  • Machine learning algorithms: For predicting CREB1 activation patterns from multi-parametric data

  • Network analysis of CREB1 target genes: To identify functional modules in specific cell types

  • Systems biology modeling: To predict CREB1 dynamics under various stimulation conditions

These emerging technologies complement traditional antibody-based detection methods by providing dynamic information that static analyses cannot capture. For instance, while phospho-specific antibodies can detect CREB1 activation at specific timepoints , real-time biosensors can reveal oscillatory patterns and cell-to-cell variability in CREB1 signaling. Similarly, proximity labeling approaches can identify novel CREB1 interaction partners that may be difficult to detect with conventional co-immunoprecipitation using anti-CREB1 antibodies.

How might CREB1 research contribute to personalized medicine approaches?

CREB1 research has significant potential to inform personalized medicine strategies across multiple disease areas:

• Stratification Biomarkers in Vaccination:

  • CREB1 activity signatures could identify individuals likely to respond to specific vaccines

  • In the RV144 HIV vaccine trial, CREB1 z-scores stratified participants into risk groups with different acquisition rates

  • Kaplan-Meier analysis showed that medium and high CREB1 z-score groups maintained significantly reduced HIV-1 acquisition risk

• Therapeutic Target in Neurological Disorders:

  • CREB1 signaling is implicated in memory formation and synaptic plasticity

  • Personalized approaches could target specific CREB1-regulated genes or upstream kinases

  • Individual variations in CREB1 pathway components could inform treatment selection

• Cancer Therapy Optimization:

  • CREB1 aberrations, like the EWSR1/CREB1 fusion in angiomatoid fibrous histiocytoma , represent targetable alterations

  • CREB1 activation patterns may predict response to specific chemotherapeutic agents

  • Combined analysis of CREB1 signaling with other pathway activities could guide precision oncology approaches

• Individualized Immune Modulation:

  • CREB1-regulated cytokine/chemokine profiles could inform personalized immunotherapy

  • Fractalkine (CX3CL1) and other CREB1 targets showed significant correlation with protection metrics in vaccine studies

  • Individual variations in CREB1 response to adjuvants could guide personalized vaccination strategies

• Pharmacogenomic Applications:

  • Genetic variations affecting CREB1 binding sites or upstream regulators may influence drug responses

  • CREB1 pathway analysis could predict individual responses to drugs targeting cAMP, Ca²⁺, or MAPK pathways

  • Patient-specific CREB1 activation profiles could guide dosing or combination therapy decisions

The identification of CREB1 as a critical driver of vaccine efficacy in HIV vaccination research exemplifies how basic molecular understanding can translate to clinically relevant stratification approaches. As technologies for monitoring CREB1 activation in clinical samples become more accessible, the potential for incorporating CREB1 biomarkers into personalized medicine algorithms will continue to grow.