CREB1 Recombinant Monoclonal Antibody

What is the biological significance of CREB1 in cellular signaling?

CREB1 functions as a nuclear transcription factor that binds to the cAMP response element (CRE) present in many viral and cellular promoters. Its activity is primarily regulated through phosphorylation at Ser133 by various receptor-activated protein kinases, including protein kinase A (PKA), calmodulin-dependent protein kinase (CaMK), and mitogen-activated protein kinases (MAPK) . CREB1 belongs to the bZIP protein family and contains one bZIP domain and one kinase-inducible domain (KID) .

Recent research has identified CREB1 as a mechanistic driver of immunogenicity in HIV-1 vaccination studies, where it mediates the recruitment of CD4+ T cells and B cells to antigen presentation sites. Significantly, CREB1 target genes include numerous cytokines and chemokines that drive immune modulatory functions .

What applications are optimal for CREB1 recombinant monoclonal antibodies?

CREB1 recombinant monoclonal antibodies demonstrate utility across multiple laboratory techniques:

Note: Optimal dilutions should be determined empirically for each experimental system .

How should samples be prepared for optimal CREB1 detection?

For phospho-CREB1 (Ser133) detection in IHC applications, antigen retrieval with TE buffer at pH 9.0 is recommended. Alternatively, citrate buffer at pH 6.0 may be used depending on tissue type and fixation method . For western blotting applications, treatment with phosphatase inhibitors (such as Calyculin A used in HEK-293 cells) is critical to preserve phosphorylation status .

For immunofluorescence applications using conjugated antibodies such as CoraLite®594-labeled CREB1 antibody, standard cell fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 provides effective results. Storage of antibody preparations should follow manufacturer guidelines, typically at -20°C with protection from light for fluorophore-conjugated antibodies .

How can researchers distinguish between total CREB1 and phosphorylated CREB1?

Distinguishing between total and phosphorylated CREB1 requires careful selection of primary antibodies. Phospho-specific antibodies like those targeting Ser133 phosphorylation will detect only the activated form of CREB1 . When designing experiments to assess CREB1 activation:

• Always run parallel samples using both phospho-specific and total CREB1 antibodies

• Calculate the ratio of phosphorylated to total CREB1 to normalize for expression differences

• Include positive controls (e.g., samples treated with cAMP elevating agents or Calyculin A)

• Include negative controls (e.g., samples treated with phosphatase)

The observed molecular weight for CREB1 typically ranges from 35-46 kDa, with phosphorylated forms often displaying slightly reduced electrophoretic mobility .

What are effective validation strategies for CREB1 antibody specificity?

Validation of CREB1 antibody specificity is critical for ensuring reliable results. Implement these methodological approaches:

• Peptide competition assays: Pre-incubate the antibody with increasing concentrations of the immunizing peptide before application to samples

• CREB1 knockdown/knockout controls: Compare staining in wild-type cells versus CREB1-depleted cells

• Multiple antibody validation: Use antibodies from different sources that recognize distinct epitopes

• Cross-reactivity testing: Test reactivity across species when working with non-human models (confirmed reactivity across human, mouse, rat, and monkey samples is noted for some CREB1 antibodies)

• Phosphorylation-specific controls: For phospho-CREB1 detection, treat samples with phosphatases to confirm signal loss

How does CREB1 antibody selection impact experimental outcomes in immunological research?

CREB1 plays a crucial role in immune responses, as evidenced by research on HIV-1 vaccination. Studies show that CREB1 target gene expression correlates with vaccine efficacy and protection from HIV-1 acquisition . When investigating CREB1 in immunological contexts:

• Consider temporal dynamics: CREB1 activity changes over time post-immunization (studies show distinct gene modulation at 16h versus 24-72h)

• Select antibodies that recognize the relevant phosphorylation site (Ser133 being crucial for activation)

• Be aware that different adjuvants may differentially impact CREB1 signaling (ALVAC+Alum versus ALVAC+MF59 showed significant differences in CREB1 target gene expression)

• Consider downstream targets: CREB1 regulates expression of cytokines and chemokines including Fractalkine (CX3CL1), GROα (CXCL1), and MCP1

What controls are essential when studying CREB1 phosphorylation dynamics?

When studying CREB1 phosphorylation dynamics, incorporate these controls:

• Time course analysis: CREB1 phosphorylation can be transient; include multiple time points

• Positive control: Samples treated with known CREB1 activators (forskolin, PMA, or BDNF)

• Inhibitor controls: Include kinase inhibitors (PKA, CaMK, MAPK inhibitors) to verify pathway specificity

• Phosphatase control: λ-phosphatase treatment to demonstrate phospho-specificity

• Loading control: Independent of CREB1 pathway to normalize for protein loading

• Subcellular localization control: Nuclear marker to confirm nuclear translocation of phosphorylated CREB1

How can researchers analyze CREB1 signaling in relation to immune function?

Systems biology approaches have validated CREB1 as a critical driver of vaccine efficacy through its regulation of immune cell recruitment and activation . Methodological approaches include:

• Transcriptional profiling: Measure expression of CREB1 target genes using RNA-seq or qPCR

• Cytokine/chemokine profiling: Quantify CREB1-regulated factors (e.g., Fractalkine, GROα, MCP1)

• Cell migration assays: Assess recruitment of immune cells (CD4+ T cells, B cells) in response to CREB1 activation

• Geneset enrichment analysis (GSEA): Identify CREB1-related transcriptional signatures

• Phospho-flow cytometry: Measure p-CREB1 levels in specific immune cell populations

Research has demonstrated that CREB1 z-scores (aggregate expression of leading-edge CREB1 target genes) correlate with reduced HIV-1 acquisition and can stratify vaccine efficacy .

What methodological approaches are recommended for investigating CREB1 in different tissue contexts?

Tissue-specific considerations for CREB1 analysis include:

• Tissue fixation: For IHC applications, suggested antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may be used for certain tissues

• Species considerations: Confirmed reactivity across human, mouse, rat, and monkey samples for selected antibodies

• Tissue-specific expression levels: Adjust antibody dilutions based on endogenous CREB1 expression (1:1000-1:4000 for IHC applications)

• Phospho-detection challenges: Fresh or rapidly frozen samples yield better results for phospho-epitopes

• Cell-type heterogeneity: Consider single-cell approaches or cell sorting when working with heterogeneous tissues

How can researchers address inconsistent CREB1 detection in Western blot applications?

Inconsistent CREB1 detection may result from several factors. Implement these methodological solutions:

• Protein extraction optimization:

  • Use RIPA or NP-40 buffer with protease and phosphatase inhibitors

  • For nuclear proteins like CREB1, ensure proper nuclear extraction

  • Maintain cold temperatures throughout extraction

• Transfer optimization:

  • For 35-46 kDa proteins like CREB1, semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 60 minutes

  • Use PVDF membranes for better protein retention

  • Verify transfer efficiency with reversible staining

• Antibody conditions:

  • The recommended dilution range of 1:5000-1:50000 for Western blot is quite broad; perform a dilution series to determine optimal concentration

  • Extend primary antibody incubation to overnight at 4°C

  • Ensure proper blocking (5% BSA is preferable for phospho-epitopes)

• Signal development:

  • Use enhanced chemiluminescence (ECL) with appropriate sensitivity

  • Avoid overexposure which may mask differences in phosphorylation

What strategies address non-specific binding in immunofluorescence applications?

For optimized CREB1 detection in immunofluorescence:

• Fixation optimization:

  • 4% paraformaldehyde for 10-15 minutes at room temperature

  • Methanol fixation may better preserve some epitopes

• Blocking optimization:

  • Extend blocking time (2 hours at room temperature)

  • Use 5-10% normal serum from the species of secondary antibody origin

  • Add 0.1-0.3% Triton X-100 for nuclear antigens like CREB1

• Antibody dilution:

  • Test multiple dilutions within the recommended range (1:50-1:500)

  • Extend incubation time (overnight at 4°C)

• Washing stringency:

  • Increase number of washes (5-6 times)

  • Add 0.05-0.1% Tween-20 to wash buffer

• Mounting media selection:

  • Use anti-fade mounting media for fluorescent dyes like CoraLite®594

  • DAPI counterstain helps confirm nuclear localization of CREB1

How does CREB1 signaling contribute to vaccine-induced immune responses?

Research demonstrates that CREB1 is a key transcriptional regulator in vaccine-induced immunity:

• Immune cell recruitment: CREB1 activation drives recruitment of CD4+ T cells and B cells to antigen presentation sites

• Cytokine/chemokine regulation: CREB1 regulates expression of immune modulatory factors:

  • Fractalkine (CX3CL1)

  • GROα (CXCL1)

  • MCP1

  • FLT3LG

  • TGF-β1/3

• Adjuvant selection impact: Different adjuvants differentially affect CREB1 activity:

  • ALVAC+Alum induces strong CREB1 target gene expression

  • ALVAC+MF59 shows significantly reduced CREB1 target gene expression

• Correlation with protection: Higher CREB1 z-scores correlate with reduced HIV-1 acquisition in vaccination trials, with participants in medium and high CREB1 z-score tertiles showing significantly lower risk

What methodological approaches are recommended for studying CREB1 in vaccine research?

Based on successful HIV-1 vaccine studies, these approaches are recommended:

• Transcriptional profiling:

  • RNA-seq of sorted immune cell populations (DCs, CD4+ T cells, B cells)

  • Analysis of CREB1 target gene expression in response to vaccination

  • Temporal analysis (16h, 24h, 72h post-vaccination) to capture dynamic responses

• Systems biology integration:

  • Gene set enrichment analysis (GSEA) to identify transcription factor activity

  • Hypergeometric means testing to verify enrichment of CREB1 target genes

  • Correlation analysis between CREB1 activity and immune outcomes

• Protein-level validation:

  • Western blot analysis of p-CREB1 (Ser133) in immune cells

  • Flow cytometry to quantify CREB1 activation in specific cell populations

  • Multiplex cytokine assays to measure downstream effectors

• In vivo validation:

  • Analysis of CREB1 target gene expression in non-human primate models

  • Correlation with protection from viral challenge

  • Assessment of antibody responses (e.g., rectal IgG against viral antigens)