Phospho-CREB1 (S133) Recombinant Monoclonal Antibody

What is the functional significance of CREB1 phosphorylation at serine 133?

Phosphorylation of CREB1 at serine 133 represents a critical activation event that enables CREB1 to function as a transcription factor. This specific phosphorylation allows CREB1 to interact with CREB binding protein (CBP), which subsequently facilitates interaction with RNA polymerase II to initiate transcription. The phosphorylation occurs in response to various stimuli including cyclic AMP, calcium, growth factors, and hormones via multiple signaling pathways including ERK1/2, PKA, PKC, and CaMKII . Once phosphorylated, CREB1 binds to cAMP-responsive elements (CRE) in the promoter regions of target genes, activating expression of proto-oncogenes such as cyclin A and Bcl-2, which regulate critical cellular processes including differentiation, proliferation, cell cycle progression, apoptosis, neovascularization, and inflammatory responses .

How do monoclonal and polyclonal Phospho-CREB1 (S133) antibodies differ in research applications?

Monoclonal and polyclonal Phospho-CREB1 (S133) antibodies offer distinct advantages depending on the experimental context:

Both antibody types exhibit specificity for the phosphorylated form of CREB1 as demonstrated in western blot validation studies with in vitro phosphorylated CREB protein, showing no reactivity to non-phosphorylated CREB . For critical quantitative research requiring highly reproducible results, the recombinant monoclonal antibody (such as clone E113) provides superior consistency and specificity for the phospho-serine 133 site .

What is the molecular weight range where Phospho-CREB1 (S133) should be detected in western blots?

While the calculated molecular weight of CREB1 is approximately 36.7 kDa, researchers should anticipate detecting Phospho-CREB1 (S133) as a band between 43-46 kDa in western blot applications. This apparent molecular weight discrepancy is consistently observed across multiple studies with validated antibodies. The observed molecular weight of approximately 52 kDa reported in some systems may reflect post-translational modifications or detection of specific isoforms . When validating new antibody lots, researchers should confirm detection specificity by comparing non-phosphorylated and phosphorylated CREB controls, as demonstrated in validation studies where antibodies show specific reactivity only to the in vitro phosphorylated form of CREB (treated with Protein Kinase A and ATP) . Additional bands at other molecular weights may represent related transcription factors with similar phosphorylation motifs or CREB isoforms resulting from alternative splicing.

What are the optimal fixation and sample preparation methods for detecting Phospho-CREB1 (S133) in different experimental systems?

The preservation of phosphorylation-specific epitopes requires careful consideration of fixation and sample preparation protocols:

For Western Blot Applications:

• Rapidly harvest samples on ice with phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate)

• Lyse cells in buffer containing 1% SDS for complete protein denaturation

• Block membranes in 5% non-fat dry milk in TBS with 0.1% Tween-20 (TBST-M) overnight at 4°C

• Use 1:500 dilution of primary antibody in TBST-M with 1-hour room temperature incubation

• Multiple washes (6× with TBST) are critical for reducing background

• Follow with appropriate HRP-conjugated secondary antibody (1:5000 dilution)

For Immunohistochemistry Applications:

• Formalin fixation and paraffin embedding preserves tissue architecture while maintaining phospho-epitope detection

• Antigen retrieval using EDTA buffer is critical for exposing the phospho-epitope

• Block endogenous peroxidase activity with 3% hydrogen peroxide for 20 minutes

• Block non-specific binding with 10% serum albumin for 30 minutes

• Use optimized antibody dilution (1:10,000 for anti-CREB1 phospho S133)

• Overnight incubation at 4°C maximizes specific binding

• Detection systems should be compatible with rabbit primary antibodies

These protocols have been validated across multiple research laboratories and provide reliable detection of authentic Phospho-CREB1 (S133) signals in diverse experimental systems .

How can phosphorylation-specific signal be validated to ensure specificity in Phospho-CREB1 (S133) detection?

Validating the phosphorylation-specific signal is critical for ensuring experimental rigor when using Phospho-CREB1 (S133) antibodies. Several complementary approaches should be employed:

• Phosphatase Treatment Controls:

  • Treat duplicate samples with lambda protein phosphatase (demonstrated effective with 2-hour treatment at 31°C)

  • Compare treated versus untreated samples to confirm signal loss in dephosphorylated samples

• In vitro Phosphorylation Controls:

  • Generate recombinant CREB protein and create paired samples: non-phosphorylated and in vitro phosphorylated using Protein Kinase A and ATP

  • Run dilution series (e.g., 100ng, 50ng, 25ng, 12.5ng) of both samples in western blots

  • Antibody should detect only the phosphorylated form across all concentrations

• Pharmacological Stimulation:

  • Treat cells with agents known to induce CREB phosphorylation (e.g., forskolin, IBMX, or (S)-3,5-DHPG)

  • Signal intensity should increase in dose-dependent manner with treatment

  • SK-N-SH cells treated with (S)-3,5-DHPG show concentration-dependent increase in Phospho-CREB1 (S133) signal

• Blocking Peptide Competition:

  • Perform dot blot analysis comparing phospho-peptide versus non-phospho peptide

  • Antibody should recognize only the phosphorylated form (as demonstrated with CREB phospho peptide)

These validation approaches collectively establish specificity for the phosphorylated serine 133 epitope and should be included in experimental design when establishing new protocols.

What are the recommended antibody dilutions and incubation conditions for different experimental techniques?

Optimal antibody dilutions and incubation conditions vary by experimental technique and should be calibrated for each specific application:

These parameters have been empirically determined from multiple studies and provide a starting point for optimization. Researchers should validate these conditions in their specific experimental systems, particularly when working with different tissue types or cell lines.

How can researchers troubleshoot weak or absent Phospho-CREB1 (S133) signal in western blots?

When encountering weak or absent signals when detecting Phospho-CREB1 (S133), consider these systematic troubleshooting approaches:

• Phosphorylation Status Verification:

  • Ensure samples contain phosphorylated CREB1 by using positive controls

  • Include freshly prepared samples from cells treated with known CREB1 activators (e.g., forskolin)

  • Measure timing of phosphorylation (often transient and peaks within 15-30 minutes)

• Sample Preparation Optimization:

  • Add phosphatase inhibitors immediately upon cell lysis (sodium fluoride at 50mM, sodium orthovanadate at 1mM)

  • Process samples rapidly to minimize dephosphorylation

  • Avoid multiple freeze-thaw cycles which can reduce phospho-epitope integrity

  • Consider using direct lysis in hot SDS-PAGE sample buffer to instantly denature phosphatases

• Technical Adjustments:

  • Increase protein loading (50-100μg total protein may be necessary)

  • For heavily glycosylated samples, consider treating with PNGase F

  • Optimize transfer conditions for higher molecular weight proteins (increase methanol content)

  • Try alternative blocking agents (BSA instead of milk for phospho-specific antibodies)

  • Extend primary antibody incubation (overnight at 4°C)

• Signal Enhancement:

  • Use higher sensitivity ECL substrates

  • Consider fluorescent secondary antibodies for quantitative analysis

  • Try biotin-streptavidin amplification systems for ultrasensitive detection

Systematic optimization of each parameter while maintaining appropriate controls will help identify the factors limiting detection in specific experimental systems.

What are the most common non-specific bands observed with Phospho-CREB1 (S133) antibodies and how can they be distinguished from authentic signal?

Non-specific bands can confound interpretation when working with Phospho-CREB1 (S133) antibodies. Here's how to identify and address common issues:

• Common Non-Specific Bands:

  • 30-35 kDa: May represent partially degraded CREB1

  • 60-65 kDa: Could be dimeric forms or cross-reactivity with related transcription factors

  • 43 kDa: Can be ATF-1, which shares homology with the phosphorylation motif

  • Multiple bands around 40-50 kDa: May represent different CREB1 splice variants

• Validation Strategies:

  • Compare with recombinant phosphorylated and non-phosphorylated CREB controls

  • Include lysates from cells where CREB1 is knocked down or knocked out

  • Pretreat samples with lambda phosphatase to eliminate phospho-specific signals

  • Use blocking peptide competition to identify specific versus non-specific bands

• Technical Approaches:

  • Increase washing stringency with higher salt concentration or detergent

  • Optimize blocking conditions (try different blocking reagents)

  • Test multiple antibody clones that recognize different phospho-epitope regions

  • Optimize primary antibody concentration (excessive antibody increases non-specific binding)

True Phospho-CREB1 (S133) signal should appear at approximately 43-46 kDa, show appropriate response to phosphorylation/dephosphorylation treatments, and exhibit expected tissue/cellular distribution patterns in control experiments .

How does sample preparation affect the stability of the Phospho-CREB1 (S133) epitope?

The labile nature of protein phosphorylation makes proper sample preparation critical for accurate Phospho-CREB1 (S133) detection:

• Timing and Temperature:

  • Process samples rapidly to minimize dephosphorylation

  • Maintain samples at 4°C during preparation

  • Avoid extended processing steps at room temperature

  • Consider snap-freezing samples in liquid nitrogen if immediate processing is not possible

• Phosphatase Inhibition Strategy:

  • Use broad-spectrum phosphatase inhibitor cocktails containing:

    • Serine/threonine phosphatase inhibitors (e.g., okadaic acid, calyculin A)

    • Tyrosine phosphatase inhibitors (e.g., sodium orthovanadate)

    • General phosphatase inhibitors (e.g., sodium fluoride, β-glycerophosphate)

  • Add inhibitors to all buffers used in sample preparation

  • Prepare inhibitor stocks fresh or store appropriately according to manufacturer recommendations

• Protein Extraction Method:

  • For western blotting, direct lysis in hot SDS-PAGE sample buffer provides immediate denaturation

  • For applications requiring native protein, use NP-40 or RIPA buffer with phosphatase inhibitors

  • For tissues, rapid homogenization in buffer containing phosphatase inhibitors is essential

  • Consider commercial phospho-protein stabilizing buffers for challenging applications

• Storage Conditions:

  • Store samples at -80°C in single-use aliquots

  • Add reducing agents (DTT or β-mercaptoethanol) immediately before use

  • Minimize freeze-thaw cycles (each cycle can reduce phospho-signal by 5-15%)

Following these guidelines ensures maximal preservation of the phosphorylated S133 epitope for accurate detection and quantification across experimental systems.

How can Phospho-CREB1 (S133) antibodies be utilized to investigate signaling pathway integration and cross-talk?

Phospho-CREB1 (S133) serves as a convergence point for multiple signaling pathways, making it an excellent tool for studying pathway integration:

• Pathway Delineation Studies:

  • Use selective inhibitors to block specific upstream kinases (PKA, PKC, CaMKII, or ERK1/2)

  • Measure changes in Phospho-CREB1 (S133) levels following pathway perturbation

  • Quantify relative contribution of each pathway to CREB1 phosphorylation under specific stimuli

  • Combine with phospho-specific antibodies for upstream kinases to map signaling cascades

• Temporal Dynamics Analysis:

  • Implement time-course experiments to capture transient versus sustained phosphorylation

  • Compare kinetics across different stimuli (e.g., growth factors versus cAMP elevating agents)

  • Correlate Phospho-CREB1 (S133) dynamics with downstream gene expression changes

  • Use pulse-chase approaches to determine CREB1 phosphorylation turnover rates

• Spatial Regulation Assessment:

  • Employ immunofluorescence to track nuclear translocation of Phospho-CREB1 (S133)

  • Perform subcellular fractionation combined with western blotting

  • Use proximity ligation assays to detect interaction with cofactors like CBP/p300

  • Implement FRET-based reporters to monitor CREB1 phosphorylation in live cells

• Stimulus Integration Models:

  • Apply combinations of stimuli activating different pathways

  • Quantify synergistic or antagonistic effects on CREB1 phosphorylation

  • Correlate with physiological or pathological outcomes in cellular models

  • Use mathematical modeling to predict pathway integration effects

These approaches leverage Phospho-CREB1 (S133) detection to reveal complex signaling relationships that regulate cellular responses to diverse stimuli .

What role does Phospho-CREB1 (S133) play in oncology research and how can antibodies against this epitope inform cancer studies?

Phospho-CREB1 (S133) has emerged as a significant biomarker in cancer research with multiple applications:

The quantitative assessment of Phospho-CREB1 (S133) levels provides valuable insights into tumor biology and patient stratification, potentially guiding personalized treatment approaches in multiple cancer types .

How can Phospho-CREB1 (S133) antibodies be used in multiplexed detection systems for comprehensive signaling analysis?

Advanced multiplexed approaches enable simultaneous assessment of Phospho-CREB1 (S133) alongside other signaling molecules:

• Multiplexed Immunofluorescence:

  • Combine Phospho-CREB1 (S133) detection with markers for:

    • Upstream kinases (phospho-PKA, phospho-ERK)

    • Related transcription factors (phospho-ATF1, phospho-CREM)

    • Downstream targets (c-Fos, BDNF)

  • Use spectrally distinct fluorophores and multispectral imaging

  • Implement tyramide signal amplification for low-abundance targets

  • Apply tissue clearing techniques for 3D visualization in thick specimens

• Mass Cytometry Approaches:

  • Label Phospho-CREB1 (S133) antibodies with rare earth metals

  • Combine with up to 40 additional markers for comprehensive signaling analysis

  • Analyze at single-cell resolution to capture population heterogeneity

  • Apply trajectory analysis to map signaling states during cellular responses

• Bead-Based Multiplexing:

  • Incorporate Phospho-CREB1 (S133) into multiplex bead arrays

  • Simultaneously quantify multiple phospho-proteins from limited samples

  • Measure dynamic changes across time points with minimal sample requirements

  • Correlate CREB1 phosphorylation with activation of parallel pathways

• Spatial Transcriptomics Integration:

  • Combine Phospho-CREB1 (S133) immunodetection with in situ transcriptomics

  • Correlate spatial patterns of CREB1 activation with target gene expression

  • Map signaling-transcription relationships in complex tissues

  • Identify microenvironmental factors influencing CREB1 activation

These multiplexed approaches provide systems-level insights into how Phospho-CREB1 (S133) functions within broader signaling networks, enabling more comprehensive understanding of complex cellular responses .

How should researchers normalize and quantify Phospho-CREB1 (S133) levels for reproducible analysis?

Proper normalization and quantification are essential for meaningful comparisons of Phospho-CREB1 (S133) across experimental conditions:

• Western Blot Quantification:

  • Always probe for total CREB1 on the same or parallel blots

  • Calculate phospho-to-total CREB1 ratio to normalize for expression differences

  • Include loading controls (β-actin, GAPDH) for sample normalization

  • Use recombinant phosphorylated CREB1 standards for absolute quantification

  • Employ linear range capture methods and analysis software for densitometry

• Immunohistochemistry Quantification:

  • Utilize optical density (OD) measurements for objective assessment

  • Capture multiple high-power fields (minimum 3) per tissue section

  • Use digital image analysis systems (e.g., Leica Qwin Standard V2.6) for consistent measurement

  • Establish threshold values based on ROC curve analysis (e.g., OD >0.28 for high expression)

  • Include control tissues on each slide for staining normalization

• Flow Cytometry Analysis:

  • Report median fluorescence intensity rather than percent positive

  • Use isotype controls and phosphatase-treated samples as negative controls

  • Calculate stimulation index (treated/untreated) for response quantification

  • Consider cell cycle phase in analysis (CREB1 phosphorylation varies by cell cycle)

• Statistical Considerations:

  • Perform replicate experiments (minimum triplicate)

  • Use appropriate statistical tests based on data distribution

  • Consider hierarchical analysis for nested experimental designs

  • Report effect sizes alongside p-values for biological relevance assessment

These standardized approaches enhance reproducibility and enable meaningful comparison of results across different experimental systems and research groups .

What are the key considerations when interpreting Phospho-CREB1 (S133) levels in disease models and patient samples?

Interpreting Phospho-CREB1 (S133) data in disease contexts requires careful consideration of multiple factors:

• Tissue-Specific Baseline Variation:

  • Normal Phospho-CREB1 (S133) levels vary significantly between tissues

  • Always compare diseased samples to matched normal tissue controls

  • Consider cell type-specific expression within heterogeneous tissues

  • Use microdissection techniques for analysis of specific cell populations when feasible

• Context-Dependent Signaling:

  • Elevated Phospho-CREB1 (S133) may have opposing effects in different cellular contexts

  • In neurons: associated with memory formation and neuroprotection

  • In cancer cells: linked to proliferation and therapy resistance

  • In inflammatory cells: mediates cytokine production and immune responses

  • Interpret changes in light of specific disease pathophysiology

• Technical Considerations in Clinical Samples:

  • Pre-analytical variables significantly impact phosphorylation status

  • Document cold ischemia time in surgical specimens

  • Consider phosphatase activity in postmortem samples

  • Standardize fixation protocols for immunohistochemistry

  • Validate antibodies specifically in the disease tissue of interest

• Correlation with Clinical Parameters:

  • Analyze associations with disease stage, grade, and patient outcomes

  • Perform multivariate analysis to identify independent prognostic value

  • Consider Phospho-CREB1 (S133) in the context of established biomarkers

  • Establish quantitative thresholds for clinical relevance

In clear cell renal cell carcinoma, for example, researchers established that high Phospho-CREB1 (S133) staining (OD >0.28) independently predicted poor outcomes even when controlling for established clinical variables, demonstrating its potential as a clinically relevant biomarker .

How do recent research findings on Phospho-CREB1 (S133) contribute to understanding mechanisms of therapy resistance?

Emerging research highlights the role of Phospho-CREB1 (S133) in therapy resistance mechanisms across multiple diseases:

• Cancer Therapy Resistance:

  • Phospho-CREB1 (S133) upregulates anti-apoptotic genes (Bcl-2, Mcl-1) reducing efficacy of cytotoxic therapies

  • Promotes metabolic adaptations supporting cancer cell survival under therapeutic stress

  • Mediates transcriptional responses to hypoxia, contributing to resistance in solid tumors

  • Activation occurs downstream of compensatory signaling pathways that emerge during targeted therapy

• Feedback Mechanisms:

  • Inhibition of specific kinases (e.g., ERK pathway) can lead to compensatory CREB1 phosphorylation via alternate pathways

  • Dual pathway inhibition strategies may prevent this compensatory activation

  • Temporal dynamics of Phospho-CREB1 (S133) can reveal adaptive responses to therapeutic intervention

  • Monitoring changes during treatment can identify emerging resistance mechanisms

• Biomarker Applications:

  • Early changes in Phospho-CREB1 (S133) levels may predict therapeutic response

  • Sequential tumor biopsies can track pathway adaptation during treatment

  • Liquid biopsy approaches may enable non-invasive monitoring

  • Combined analysis with other phospho-proteins provides comprehensive resistance profiling

• Therapeutic Vulnerabilities:

  • CREB1-dependent cells may exhibit synthetic lethality with specific pathway inhibitors

  • Targeting cofactors required for CREB1 transcriptional activity may overcome resistance

  • Combination strategies blocking both CREB1 phosphorylation and function show enhanced efficacy

  • Pulsatile treatment schedules may prevent adaptation and resistance development

Understanding the dynamic role of Phospho-CREB1 (S133) in therapy resistance provides opportunities for developing more effective treatment strategies and implementing rational drug combinations based on molecular mechanisms rather than empirical testing.