Phospho-CREB1 (T100) Antibody

What is Phospho-CREB1 (T100) Antibody and what does it specifically detect?

Phospho-CREB1 (T100) Antibody is a rabbit polyclonal antibody that specifically detects endogenous levels of CREB1 protein only when phosphorylated at threonine 100 (T100) . This antibody recognizes the phosphorylated form of Cyclic AMP-responsive element-binding protein 1 (CREB1), which functions as a phosphorylation-dependent transcription factor that stimulates transcription upon binding to the DNA cAMP response element (CRE) . The specificity for the phosphorylated form makes this antibody particularly valuable for studying CREB1 activation states in various cellular contexts .

What species reactivity does Phospho-CREB1 (T100) Antibody demonstrate?

Phospho-CREB1 (T100) Antibody demonstrates reactivity across multiple mammalian species including human, mouse, and rat samples . This cross-species reactivity makes the antibody particularly valuable for comparative studies and allows researchers to use various model systems while maintaining consistent detection methodologies. When designing experiments with new cell lines or tissue types, preliminary validation is still recommended despite the broad reactivity profile .

What are the recommended applications for Phospho-CREB1 (T100) Antibody?

The primary validated applications for Phospho-CREB1 (T100) Antibody include Western Blot (WB) and ELISA . For Western Blot applications, the recommended dilution range is 1:500-1:2000, which should be optimized based on sample type and detection method . For ELISA applications, a significantly higher dilution of 1:20000 is typically recommended . The antibody has been rigorously validated for these specific applications through experimental testing with appropriate controls to ensure reliable and reproducible results .

How should Phospho-CREB1 (T100) Antibody be stored for optimal stability?

For optimal stability and performance, Phospho-CREB1 (T100) Antibody should be stored at -20°C or -80°C upon receipt . The antibody is typically formulated in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide, which helps maintain stability during storage . It is important to avoid repeated freeze-thaw cycles as these can degrade antibody performance . The antibody can generally be stored for up to one year from the date of receipt when stored properly . Aliquoting the antibody into smaller volumes before freezing can help minimize freeze-thaw cycles when the entire volume is not needed at once.

How does phosphorylation at T100 differ functionally from other CREB1 phosphorylation sites like S133?

While CREB1 phosphorylation at S133 is well-characterized and associated with transcriptional activation, phosphorylation at T100 represents a distinct regulatory mechanism . CREB1 functions as a phosphorylation-dependent transcription factor involved in multiple cellular processes including circadian rhythm synchronization, adipose cell differentiation, and regulation of apoptotic and inflammatory responses in cardiomyocytes . Unlike S133 phosphorylation which is primarily mediated by PKA, T100 phosphorylation may engage different upstream kinases and signaling pathways . When designing experiments to study CREB1 regulation, researchers should consider using both T100 and S133 phospho-specific antibodies to gain comprehensive insights into the dynamic regulation of this transcription factor under various stimuli and conditions.

What controls should be included when validating Phospho-CREB1 (T100) Antibody specificity in new experimental systems?

For rigorous validation of Phospho-CREB1 (T100) Antibody in new experimental systems, several controls should be implemented. First, include both phosphatase-treated samples (to eliminate the phospho-epitope) and stimulated samples (to induce T100 phosphorylation) . Second, utilize competing peptides containing the phosphorylated and non-phosphorylated forms of the T100 site to confirm epitope specificity . Third, consider using CREB1 knockout or knockdown samples as negative controls . Fourth, compare results with a different phospho-independent CREB1 antibody to verify total protein levels . Finally, include positive control samples from previously validated cell types or tissues that are known to express phosphorylated CREB1 at T100 .

What are the optimal cell lysis and sample preparation methods for detecting phosphorylated CREB1 at T100?

For optimal detection of phosphorylated CREB1 at T100, cell lysis and sample preparation procedures must carefully preserve phospho-epitopes. The recommended protocol includes: (1) rapid harvesting of samples in the presence of phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, and phosphatase inhibitor cocktails); (2) lysis in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, supplemented with protease and phosphatase inhibitors ; (3) brief sonication to shear genomic DNA; (4) centrifugation at 14,000 × g for 15 minutes at 4°C to clear cellular debris; and (5) immediate addition of SDS sample buffer and heating to 95°C for 5 minutes . For nuclear proteins like CREB1, consider using specialized nuclear extraction protocols to enrich for the target protein. Fresh samples typically yield better results than frozen ones when analyzing phosphorylation states.

What stimulation conditions effectively induce CREB1 phosphorylation at T100 for positive control samples?

To generate reliable positive control samples with phosphorylated CREB1 at T100, several stimulation conditions have proven effective across different cell types. Treatment with forskolin (10 μM for 30 minutes), which activates adenylyl cyclase and increases intracellular cAMP levels, can induce CREB1 phosphorylation . Alternative approaches include phorbol esters like PMA (100 nM for 15-30 minutes), growth factors like EGF or BDNF (50 ng/mL for 15 minutes), or calcium ionophores such as ionomycin (1 μM for 30 minutes) . For neuronal cells, glutamate receptor activation (50 μM glutamate for 5 minutes) can effectively induce CREB1 phosphorylation. When establishing positive controls, time-course experiments are recommended as phosphorylation events can be transient, and optimal stimulation times may vary by cell type and stimulus .

How can researchers quantitatively analyze changes in CREB1 T100 phosphorylation in complex experimental designs?

Quantitative analysis of CREB1 T100 phosphorylation requires rigorous methodological approaches, especially in complex experimental designs. For Western blot applications, densitometric analysis should normalize phospho-CREB1 (T100) signals to total CREB1 protein levels from parallel blots or after membrane stripping and reprobing . For multi-condition experiments, consider using multiplexed detection systems that allow simultaneous analysis of phosphorylated and total protein forms. ELISA-based quantification can provide more precise numerical data, with the antibody used at a 1:20000 dilution for optimal sensitivity and specificity . For spatial analysis of phosphorylation in heterogeneous samples, consider phospho-specific immunohistochemistry followed by digital image analysis. In all quantitative applications, standard curves using recombinant phosphorylated proteins can enhance accuracy, and statistical analysis should account for biological and technical replicates.

What methodological approaches can resolve contradictory results between phospho-specific and total CREB1 antibody signals?

When facing contradictory results between phospho-specific and total CREB1 antibody signals, systematic troubleshooting and methodological refinements are necessary. First, verify antibody specificity using competing peptides (phosphorylated versus non-phosphorylated) to confirm that the phospho-antibody is truly detecting the modified form . Second, investigate potential phosphatase activity during sample preparation by comparing fresh lysates with those prepared with enhanced phosphatase inhibitor cocktails . Third, evaluate whether the epitope recognized by the total CREB1 antibody might be masked by protein-protein interactions or additional post-translational modifications that could affect binding . Fourth, consider that rapid turnover of the phosphorylated form might lead to apparent discrepancies in signal intensity. Finally, employing alternative detection methods such as Phos-tag SDS-PAGE, which can separate phosphorylated from non-phosphorylated proteins based on mobility shifts, might provide clarification when standard approaches yield contradictory results .

How can Phospho-CREB1 (T100) Antibody be effectively incorporated into multiplexed phospho-protein analysis workflows?

Incorporating Phospho-CREB1 (T100) Antibody into multiplexed phospho-protein analysis requires careful optimization of several technical parameters. For fluorescence-based multiplexed Western blotting, this rabbit polyclonal antibody can be paired with mouse monoclonal antibodies targeting other phospho-proteins of interest, using species-specific secondary antibodies with distinct fluorophores . When designing such experiments, consider potential cross-reactivity between secondary antibodies and ensure adequate spectral separation between fluorophores. For bead-based multiplexed assays, the antibody can be conjugated to capture beads following manufacturer protocols, with optimization of antibody concentration crucial for maintaining specificity . In cell-based multiplexed assays, careful selection of additional antibodies raised in different host species is essential. When multiplexing phospho-specific antibodies, synchronizing cellular stimulation to capture phosphorylation events that occur with different kinetics may require careful experimental timing or the use of phosphatase inhibitors to stabilize transient modifications .

How can Phospho-CREB1 (T100) Antibody be optimized for use in chromatin immunoprecipitation (ChIP) experiments?

While Phospho-CREB1 (T100) Antibody is primarily validated for Western blot and ELISA applications , adapting it for chromatin immunoprecipitation (ChIP) requires specific optimization strategies. First, crosslinking conditions should be carefully titrated, as excessive fixation may mask the phospho-epitope. A recommended starting point is 1% formaldehyde for 10 minutes at room temperature. Second, sonication conditions should be optimized to generate DNA fragments of 200-500 bp while preserving antibody epitopes. Third, use a modified RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, and both protease and phosphatase inhibitors for immunoprecipitation . Fourth, increase the antibody concentration compared to Western blot applications, starting with 5-10 μg per ChIP reaction. Finally, include appropriate controls such as non-phosphorylated CREB1 ChIP and IgG controls. Validating ChIP results by examining enrichment at known CREB1 target genes containing cAMP response elements (CRE) is essential for confirming the specificity of phospho-CREB1 binding events.

What considerations are important when using Phospho-CREB1 (T100) Antibody in primary neuronal culture experiments?

When utilizing Phospho-CREB1 (T100) Antibody in primary neuronal culture experiments, several specialized considerations are important. First, CREB1 plays critical roles in neuronal function, including synaptic plasticity, memory formation, and neuronal survival, making phosphorylation status particularly relevant in neuronal contexts . Second, basal phosphorylation levels may vary significantly depending on neuronal activity states, so carefully controlled culture conditions are essential. Third, when stimulating neurons to induce phosphorylation, consider physiologically relevant stimuli such as BDNF (50 ng/mL), glutamate receptor activation, or KCl-induced depolarization (50 mM for 15 minutes) . Fourth, subcellular localization of phosphorylated CREB1 is particularly important in neurons, so consider complementing Western blot analysis with immunocytochemistry to visualize nuclear translocation. Finally, when analyzing regional differences in CREB1 phosphorylation within complex neuronal structures, microdissection followed by Western blotting may provide more informative results than whole-culture lysates.

How does CREB1 T100 phosphorylation interact with other post-translational modifications of the protein?

CREB1 undergoes multiple post-translational modifications that can interact with T100 phosphorylation in complex regulatory networks. While Ser133 phosphorylation is the most well-characterized CREB1 modification associated with transcriptional activation, T100 phosphorylation represents a distinct regulatory mechanism that may work cooperatively or antagonistically with other modifications . Studies suggest that phosphorylation patterns can function as a molecular barcode, with different combinations of modifications leading to recruitment of specific cofactor complexes. Beyond phosphorylation, CREB1 can undergo acetylation, SUMOylation, glycosylation, and ubiquitination, each potentially influencing protein stability, localization, or function . When investigating these interactions, consider using phospho-specific antibodies against multiple sites (such as both T100 and S133) in parallel experiments . Mass spectrometry-based approaches can also provide comprehensive mapping of modification patterns in different cellular contexts and under various stimuli.

What technical adaptations are needed for using Phospho-CREB1 (T100) Antibody in formalin-fixed paraffin-embedded (FFPE) tissue samples?

While Phospho-CREB1 (T100) Antibody is primarily validated for Western blot and ELISA applications , adapting it for use with formalin-fixed paraffin-embedded (FFPE) tissue samples requires several technical modifications. First, antigen retrieval is critical for exposing the phospho-epitope, with citrate buffer (pH 6.0) heated to 95-100°C for 20 minutes as a recommended starting point. Second, include a peroxidase quenching step (3% hydrogen peroxide for 10 minutes) before blocking to reduce background. Third, use a more concentrated antibody dilution than for Western blot, starting at 1:50-1:200, and optimize with titration experiments . Fourth, extend primary antibody incubation times to overnight at 4°C to enhance sensitivity. Fifth, include phosphatase inhibitors in all buffers to prevent dephosphorylation during processing. Finally, validate specificity using appropriate controls including peptide competition with phosphorylated and non-phosphorylated peptides. Comparison with frozen tissue sections from the same source can help establish whether FFPE processing affects epitope recognition by this phospho-specific antibody.

How can Phospho-CREB1 (T100) Antibody be utilized in high-throughput drug screening platforms?

Incorporating Phospho-CREB1 (T100) Antibody into high-throughput drug screening platforms offers potential for identifying compounds that modulate CREB1 signaling pathways. For cell-based assays, optimize the antibody for in-cell Western or high-content imaging platforms using appropriate secondary antibodies or direct conjugates . The recommended starting dilution would be 1:500, with optimization necessary for specific detection platforms . Develop a robust positive control using forskolin or other CREB1-activating compounds to establish signal windows and Z-factors for assay validation . Miniaturization to 384- or 1536-well formats requires careful optimization of cell density, fixation protocols, and antibody concentrations. For automated image-based screening, nuclear translocation of phosphorylated CREB1 can serve as a primary readout, quantified through nuclear/cytoplasmic intensity ratios. Multiplexing with markers of cell viability and total CREB1 levels can enhance screening specificity and reduce false positives. Validation of hit compounds should include dose-response analyses and orthogonal assays to confirm mechanism of action.

What emerging single-cell analysis methods can benefit from Phospho-CREB1 (T100) Antibody application?

Emerging single-cell analysis technologies offer new opportunities for applying Phospho-CREB1 (T100) Antibody to understand cellular heterogeneity in CREB1 signaling. For mass cytometry (CyTOF) applications, the antibody can be metal-conjugated according to standard protocols, allowing integration into larger phospho-protein panels to examine signaling network relationships at single-cell resolution . In imaging mass cytometry, the antibody can reveal spatial distribution of T100 phosphorylation in tissue contexts. For microfluidic-based single-cell Western blotting, the antibody can be used at a 1:500 dilution, with optimization for reduced sample volumes . Single-cell phospho-proteomics workflows may incorporate the antibody in targeted enrichment strategies before mass spectrometry analysis. When adapting for these advanced applications, careful validation of specificity in the modified format is essential, comparing results with conventional bulk analysis methods. These approaches can reveal previously undetectable subpopulations of cells with distinct CREB1 phosphorylation states that might be masked in population-averaged measurements.

How do different cellular stressors affect CREB1 T100 phosphorylation patterns compared to other phosphorylation sites?

Different cellular stressors can induce distinct patterns of CREB1 phosphorylation across various sites including T100. Oxidative stress (H₂O₂ treatment), nutrient deprivation, hypoxia, DNA damage, and endoplasmic reticulum stress each engage different upstream kinase cascades that may differentially phosphorylate CREB1 at T100 versus other sites like S133 . Using Phospho-CREB1 (T100) Antibody in parallel with antibodies targeting other phosphorylation sites can reveal stress-specific phosphorylation signatures . The temporal dynamics of phosphorylation often differ between sites, with some modifications serving as rapid, transient signals while others persist longer and mediate sustained transcriptional responses . When investigating stress responses, consider time-course experiments spanning minutes to hours to capture both immediate and delayed phosphorylation events. Quantitative analysis should include normalization to total CREB1 levels and comparison across multiple phosphorylation sites to develop a comprehensive understanding of how specific stressors reprogram CREB1-dependent transcriptional networks through combinatorial phosphorylation patterns.

What are the methodological approaches for studying CREB1 T100 phosphorylation in patient-derived samples for translational research?

Studying CREB1 T100 phosphorylation in patient-derived samples presents unique challenges requiring specialized methodological approaches. For fresh tissue biopsies, rapid processing is critical to preserve phosphorylation status, ideally flash-freezing samples in liquid nitrogen within minutes of collection . For surgical specimens, document cold ischemia time as phosphorylation status may change rapidly post-excision. When working with banked biospecimens, evaluate phospho-epitope stability under the specific storage conditions used. For patient-derived cell lines or primary cultures, standardize culture conditions and passage numbers to minimize variability . When analyzing clinical samples by Western blot, include matched normal tissues or reference standards to enable cross-sample comparisons, using the recommended antibody dilution of 1:500-1:2000 . For tissue microarrays, optimize immunohistochemistry protocols with phosphatase inhibitors and appropriate controls. Finally, correlate phosphorylation data with clinical parameters and outcomes using appropriate statistical methods for biomarker analysis, considering the potential heterogeneity within patient populations and the need for adequate sample sizes to achieve statistical power.