CREB1 Antibody

What criteria should be considered when selecting a CREB1 antibody for experimental applications?

When selecting a CREB1 antibody, researchers should consider:

• Target epitope: Antibodies targeting different domains (e.g., bZIP DNA-binding domain, dimerization domain, or kinase inducible domain) may yield different results. For instance, antibodies targeting amino acids 254-327 within the DNA binding and dimerization domain can detect both CREB-1A and CREB-1B isoforms .

• Host species: Consider the compatibility with your experimental design, especially for co-staining experiments. Mouse monoclonal IgG2a and IgG2b are commonly used .

• Cross-reactivity: Verify reactivity with your species of interest. Many CREB1 antibodies react with human, mouse, and rat samples, while some also detect equine, canine, bovine, and porcine CREB1 .

• Detection of phosphorylated forms: For studying CREB1 activation, specific antibodies against phosphorylated Ser133 are critical .

• Validated applications: Confirm the antibody has been validated for your specific application (WB, IHC, IF, IP, ChIP) .

How can I distinguish between total CREB1 and phosphorylated CREB1 in my experiments?

Distinguishing between total and phosphorylated CREB1 requires:

• Antibody selection: Use antibodies specifically raised against phosphorylated CREB1 at Ser133, such as phospho-specific antibodies that recognize this modification .

• Parallel detection: Run parallel experiments with antibodies against total CREB1 (tCREB1) and phosphorylated CREB1 (pCREB1) to determine activation status relative to total protein levels .

• Dephosphorylation controls: Include samples treated with phosphatases to confirm specificity of phospho-antibodies.

• Molecular weight confirmation: Phosphorylated CREB1 typically appears at approximately 43 kDa in western blots, but observed weights may vary (43-46 kDa or 52 kDa have been reported) .

• Time course analysis: Consider temporal dynamics of phosphorylation - Liu et al. demonstrated that CREB1 levels increased in two phases following stimulation, with peaks at 2h and then again at 18-24h .

For quantitative analysis, researchers often calculate the ratio of phosphorylated to total CREB1 to normalize for variations in protein loading or expression levels.

What are the optimal protocols for using CREB1 antibodies in western blotting?

For successful western blotting with CREB1 antibodies:

Sample preparation:

• Use phosphatase inhibitors in lysis buffers when detecting phosphorylated CREB1

• Recommended concentration: 1.0 µg/mL for western blotting

Protocol considerations:

• Running conditions: Use reducing conditions with appropriate buffer systems (e.g., Immunoblot Buffer Group 3)

• Transfer: PVDF membrane is commonly used for CREB1 detection

• Blocking: Typically 5% non-fat milk or BSA (BSA preferred for phospho-antibodies)

• Primary antibody incubation: Typically overnight at 4°C

• Detection: Both chemiluminescence and fluorescence detection are suitable

Expected results:

• CREB1 appears at approximately 43 kDa

• CREB1 is primarily detected in nuclear fractions

• Phosphorylated CREB1 (p-CREB1) may show temporal variations depending on stimulation conditions

Validation:

• Include positive controls (cell lines with known CREB1 expression like A20 mouse B cell lymphoma)

• Include negative controls (CREB1 knockdown samples or cells known to lack CREB1)

• Include MW markers to confirm band identity

How can I optimize immunohistochemistry and immunofluorescence protocols for CREB1 detection?

For optimal IHC/IF detection of CREB1:

Tissue preparation:

• Formalin-fixed paraffin-embedded (FFPE) tissues work well with many CREB1 antibodies

• For immunofluorescence, fixation in 4% paraformaldehyde with 30% sucrose is effective

Antigen retrieval:

• Heat-induced epitope retrieval in citrate buffer (pH 6.0) is commonly used

• For phospho-CREB1 detection, phosphatase inhibitors should be included in buffers

Antibody dilutions:

• For IHC-P: Recommended concentration range of 1.0-5.0 µg/ml for monoclonal antibodies

• For polyclonal antibodies: Dilutions of 1:25-1:100 may be appropriate

Detection systems:

• For immunofluorescence: Secondary antibodies conjugated to fluorophores (e.g., cyanine 3)

• For IHC: HRP-conjugated secondary antibodies with DAB or AEC substrate

Analysis approaches:

• Confocal microscopy with z-series of optical sections (0.5 μm increments) allows precise nuclear localization

• MetaMorph Offline software can be used for quantification of mean fluorescence intensity

• Sample size: 5-10 neurons per dish is often sufficient for quantitative analysis

What are the key considerations for using CREB1 antibodies in ChIP and ChIP-seq experiments?

For effective chromatin immunoprecipitation with CREB1 antibodies:

Antibody selection:

• Use ChIP-validated antibodies that specifically recognize the DNA-binding domain

• Both monoclonal and polyclonal antibodies can work for ChIP, but validation is essential

Experimental design:

• Include appropriate controls: IgG control, input DNA control, and positive controls (known CREB1 target genes)

• Consider crosslinking conditions: standard 1% formaldehyde for 10 minutes works well for transcription factors

Protocol optimization:

• Sonication conditions should be optimized to generate 200-500bp DNA fragments

• ChIP-qPCR validation should precede ChIP-seq to confirm enrichment of known targets

• CREB1 binds the cAMP response element (CRE) with the consensus sequence TGACGTCA

Data analysis:

• Analysis should focus on identifying CREB1 binding sites containing the CRE motif

• CREB1 target genes include cytokines/chemokines that are important for immune responses

• The GSEA (Gene Set Enrichment Analysis) approach can be used to identify CREB1 target gene sets

How can CREB1 antibodies be used to study its role in immunity and vaccine development?

CREB1 has emerged as a critical factor in vaccine efficacy, particularly in HIV-1 vaccine development:

Experimental approaches:

• Transcriptional profiling of purified immune cells (DCs, CD4+ T cells, B cells) can be performed with CREB1 antibodies to assess activation status

• Flow cytometry with phospho-CREB1 antibodies can identify activated immune cell populations

• ChIP-seq using CREB1 antibodies can identify target genes involved in immune responses

Key findings from HIV vaccine research:

• Induction of CREB1 and its target genes by the ALVAC vector correlates with reduced HIV-1 acquisition in clinical trials

• CREB1-regulated genes include cytokines/chemokines associated with protection from SIV challenge in non-human primates

• CREB1 activity drives recruitment of CD4+ T cells and B cells to the site of antigen presentation

Analytical approaches:

• CREB1 z-scores can be calculated from the expression of CREB1 target genes

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

• CREB1 target genes showed significant positive enrichment for genes correlating with protective antibody responses

This research demonstrates how CREB1 antibodies can be powerful tools for understanding immune mechanisms in vaccine development.

What are the approaches for studying temporal dynamics of CREB1 expression and phosphorylation?

Studying CREB1 temporal dynamics requires careful experimental design:

Time course experiments:

• CREB1 levels can increase in biphasic patterns after stimulation: initial increase at 2h, return to baseline at 12h, and second increase at 18-24h post-stimulation

• Phosphorylation at Ser133 may follow different kinetics than total protein levels

Methodological approaches:

• Immunofluorescence with confocal microscopy allows quantification of CREB1 levels in specific cellular compartments over time

• Western blotting with phospho-specific and total CREB1 antibodies at multiple timepoints

• Live-cell imaging with fluorescently tagged CREB1 constructs can complement antibody-based approaches

Data from temporal studies:

• In neuronal studies, 5-HT treatment increased CREB1 levels to 150±6% of control at 2h, returned to baseline at 12h, then increased again to 132±7% at 18h and 128±3% at 24h

• Nuclear CREB1 follows similar patterns: 153±19% at 2h, 99±15% at 12h, 134±9% at 18h, and 130±5% at 24h

• In vaccine studies, CREB1 target gene expression was downregulated at 16h but significantly enhanced at 24h and continued to increase up to 72h after vaccination

These approaches reveal how CREB1 regulation occurs in waves that may correspond to different phases of cellular responses.

How can I use CREB1 antibodies to investigate its role in disease models?

CREB1 has been implicated in various diseases, and antibodies can be valuable tools for mechanistic studies:

Cancer research applications:

• CREB1 levels correlate with cancer progression in multiple cancer types

• Elevated CREB1 has been associated with leukemia, lymphoma, melanoma, and various solid tumors

• CREB1 inhibition promotes anti-tumoral immunity by limiting HLA-E expression and enhancing NK cell activity

Methodological approaches:

• Tissue microarrays with CREB1 immunohistochemistry can assess expression across tumor samples

• Western blotting can quantify CREB1 and phospho-CREB1 levels in patient samples

• siRNA knockdown followed by CREB1 antibody detection can confirm target specificity

Periodontitis models:

• Zoledronic acid (ZA) treatment of periodontal ligament stem cells (PDLSCs) leads to decreased CREB1 expression

• CREB1 overexpression alleviates apoptosis and enhances viability in ZA-challenged PDLSCs

• CREB1 regulates VEGF expression through direct binding to its promoter, demonstrated through ChIP assays

Neurodegenerative disease applications:

• CREB1 plays crucial roles in neuronal survival and memory formation

• Phospho-CREB1 levels can be used as markers of neuronal activity and plasticity

• Decreased CREB1 function has been implicated in cognitive disorders

What are common challenges and solutions when working with CREB1 antibodies?

Researchers often encounter several challenges when working with CREB1 antibodies:

Challenge: Cross-reactivity with related proteins

• CREB1 shares homology with CREM and ATF-1

• Solution: Select antibodies validated for specificity, particularly monoclonal antibodies targeting unique epitopes

• Some antibodies (e.g., D-12 clone) detect CREB-1A, CREB-1B, CREM, and ATF-1 isoforms, which may be advantageous or problematic depending on your research question

Challenge: Phosphorylation-dependent epitope masking

• Some antibodies may have reduced binding to phosphorylated CREB1

• Solution: Use antibodies specifically validated for detecting total CREB1 regardless of phosphorylation state

Challenge: Subcellular localization analysis

• CREB1 is predominantly nuclear but can be present in cytoplasm

• Solution: Nuclear/cytoplasmic fractionation or confocal microscopy with z-stack analysis can help resolve localization

Challenge: Low signal in western blots

• Solution: Optimize protein extraction methods (CREB1 is a nuclear protein), increase antibody concentration, extend incubation time, and consider enhanced detection systems

Challenge: Inconsistent immunohistochemistry results

• Solution: Optimize fixation and antigen retrieval methods; for FFPE samples, a concentration range of 1.0-5.0 µg/ml is recommended for IHC-P

How can I validate the specificity of my CREB1 antibody results?

Proper validation is crucial for ensuring reliable CREB1 antibody results:

Positive controls:

• Use cell lines with known CREB1 expression (e.g., A20 mouse B cell lymphoma, M1 mouse myeloid leukemia)

• Include samples with stimulated CREB1 phosphorylation (e.g., cAMP pathway activators)

Negative controls:

• CREB1 knockdown via siRNA (as demonstrated in Liu et al., where CREB1 siRNA blocked the 5-HT-induced increase in CREB1)

• Genetic knockout models where available

• Peptide competition assays to confirm epitope specificity

Orthogonal methods:

• Compare results with multiple CREB1 antibodies targeting different epitopes

• Confirm key findings with non-antibody methods (e.g., CRISPR-Cas9 editing, reporter assays)

Phosphorylation validation:

• For phospho-CREB1 antibodies, include samples treated with phosphatases

• Include appropriate stimulation controls (e.g., forskolin treatment increases CREB1 phosphorylation)

Molecular weight confirmation:

• CREB1 typically appears at 43-46 kDa (observed) despite a calculated molecular weight of 35-36 kDa

• Some phosphorylated forms may appear at 52 kDa

How can CREB1 antibodies be incorporated into single-cell analysis workflows?

Integrating CREB1 antibodies into single-cell technologies offers new research possibilities:

Single-cell immunofluorescence:

• CREB1 and phospho-CREB1 antibodies can be used for high-content imaging to assess heterogeneity in cellular responses

• Cell-to-cell variability in CREB1 activation can be quantified (as demonstrated in neuronal studies examining 5-10 neurons per experimental condition)

Mass cytometry (CyTOF):

• Metal-conjugated CREB1 antibodies can be incorporated into CyTOF panels

• Enables simultaneous detection of CREB1 with multiple surface and intracellular markers

• Particularly valuable for immune cell phenotyping in conjunction with CREB1 activation status

Single-cell western blotting:

• Emerging technologies allow protein detection at single-cell resolution

• CREB1 and phospho-CREB1 antibodies can be used to examine activation at the individual cell level

Spatial transcriptomics integration:

• CREB1 antibodies can be combined with in situ hybridization techniques

• Allows correlation between CREB1 protein levels/activation and spatial gene expression patterns

• Particularly relevant given CREB1's role in regulating specific gene sets in immune responses

What are future directions for CREB1 antibody applications in research?

Emerging research directions using CREB1 antibodies include:

Therapeutic development monitoring:

• CREB1 antibodies can assess the efficacy of CREB1 inhibitors being developed for cancer treatment

• Changes in CREB1 phosphorylation can serve as pharmacodynamic biomarkers

Multi-omics integration:

• Combining ChIP-seq using CREB1 antibodies with RNA-seq and proteomics to build comprehensive regulatory networks

• Using CREB1 z-scores derived from target gene expression as predictive biomarkers, as demonstrated in HIV vaccine research

Extracellular vesicle analysis:

• Examining CREB1 and its targets in extracellular vesicles as potential biomarkers

• CREB1 may regulate genes involved in vesicle production and content

Advanced imaging:

• Super-resolution microscopy with CREB1 antibodies to examine nuclear organization of transcription factories

• Multiplexed imaging techniques to examine CREB1 in relation to multiple pathway components simultaneously

Gene therapy monitoring:

• CREB1 antibodies can be used to monitor transgene expression and function in gene therapy approaches targeting CREB1-dependent pathways