CREB1 Antibody Pair

What criteria should be considered when selecting a CREB1 antibody pair for sandwich ELISA?

When selecting antibody pairs for CREB1 sandwich ELISA, researchers should consider:

• Epitope complementarity: Select antibodies that bind to distinct, non-overlapping epitopes on CREB1

• Species reactivity: Confirm reactivity with your species of interest (human, mouse, rat)

• Phosphorylation specificity: Determine if you need antibodies that differentiate between phosphorylated and non-phosphorylated forms

• Isoform detection: Consider which CREB1 isoform(s) need to be detected

• Validation data: Review published validation data including Western blot, IHC, and ELISA results

CREB1 contains multiple phosphorylation sites that affect its function, including Ser133, Ser129, and Ser142, making antibody selection particularly important for functional studies .

How do I validate a new CREB1 antibody pair before implementing it in my research?

A systematic validation approach should include:

• Positive and negative controls: Use cell lines or tissues known to express or lack CREB1

• Cross-reactivity testing: Test against related proteins (CREM, ATF-1)

• Specificity validation: Use siRNA knockdown of CREB1 to confirm signal reduction

• Phosphorylation induction: For phospho-specific antibodies, induce phosphorylation with PKA activators and verify with Western blotting

• Dynamic range determination: Generate a standard curve using recombinant CREB1

For example, studies have validated CREB1 antibody specificity by demonstrating reduced RRM2 protein levels following CREB1 knockdown in multiple CRC cell lines, confirming antibody specificity and target engagement .

What are the differences between total CREB1 and phosphorylated CREB1 antibodies, and when should each be used?

Phosphorylated forms indicate activated CREB1, with Ser133 phosphorylation being particularly critical for transcriptional activity. Phospho-Ser133 antibodies are extensively used in studies examining pathway activation, as seen in AA-resistant cell line research where phosphorylated CREB1 (pCREB1) levels correlate with resistance mechanisms .

How can I effectively use CREB1 antibody pairs to measure phosphorylation dynamics in time-course experiments?

For time-course experiments measuring CREB1 phosphorylation dynamics:

• Dual antibody approach: Use a pair consisting of phospho-specific and total CREB1 antibodies

• Normalization strategy: Express phospho-CREB1 signal relative to total CREB1 to account for expression changes

• Temporal resolution: Consider short intervals (minutes) for initial phosphorylation and longer intervals (hours) for sustained effects

• Quantification method: Use densitometry for Western blots or mean fluorescence intensity for flow cytometry

Example time points from published research show CREB1 target gene signatures are downregulated at 16 hours but significantly enhanced at 24 hours and continue to increase up to 72 hours after ALVAC vaccination, demonstrating the importance of proper temporal analysis .

What controls should be included when using CREB1 antibody pairs in ChIP-seq experiments?

For ChIP-seq experiments using CREB1 antibody pairs:

• Input control: Chromatin before immunoprecipitation

• IgG control: Non-specific IgG from the same species as the CREB1 antibody

• Positive control loci: Known CREB1 binding sites (e.g., CRE motif 5′-TGACGTCA-3′)

• Negative control loci: Genomic regions not bound by CREB1

• Knockdown/knockout validation: CREB1-depleted samples

• PhosphoSTOP inhibitors: Include in lysis buffers to preserve phosphorylation status

Research has demonstrated that CREB1 directly binds to specific promoter regions, as shown in chromosome immunoprecipitation (ChIP) assays that confirmed CREB1 binding to the RRM2 promoter region around CRE-site2 (-1039/-1032) .

How do I distinguish between CREB1 isoforms using antibody-based techniques?

CREB1 has multiple isoforms that can be differentiated using:

• Isoform-specific antibodies: Target unique regions or splice junctions

• Western blot analysis: Distinguish based on molecular weight differences (observed 37-46 kDa depending on isoform)

• RT-PCR validation: Use isoform-specific primers in conjunction with antibody detection

• Immunoprecipitation followed by mass spectrometry: For detailed isoform characterization

Primer design is critical—select regions specific to each isoform to avoid cross-reactions. For example, real-time PCR studies successfully differentiated LymCREB1 isoforms by designing primer sets in specific regions of each isoform, with cross-reactant concentrations consistently below one-thousandth of the target PCR product concentrations .

How can I use CREB1 antibody pairs to investigate protein-protein interactions in the transcriptional complex?

For studying CREB1 protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use a CREB1 antibody to pull down the complex

  • Probe with antibodies against suspected interaction partners (CBP/p300, TORC)

  • Include phosphatase inhibitors to preserve phosphorylation-dependent interactions

• Proximity Ligation Assay (PLA):

  • Use two primary antibodies (CREB1 + partner protein)

  • PLA signals indicate proteins within 40nm proximity

  • Quantify interaction frequency and subcellular localization

• Sequential ChIP (Re-ChIP):

  • First IP with CREB1 antibody

  • Second IP with antibody against interaction partner

  • Identifies co-occupancy at specific genomic loci

Research has shown that CREB1-CBP/p300 interactions are critical in transcriptional regulation, with studies demonstrating that AA-resistant cells exhibit higher sensitivity to CBP/p300 inhibitors, suggesting enhanced CREB1-CBP/p300 activity in these cells .

What methodological approaches can resolve contradictory results between phosphorylated CREB1 levels and transcriptional activity?

When phospho-CREB1 levels don't correlate with expected transcriptional activity:

• Multi-site phosphorylation analysis:

  • Use antibodies against different phosphorylation sites (Ser133, Ser129, Ser142)

  • Certain modifications may have inhibitory effects

• Subcellular fractionation:

  • Separate nuclear and cytoplasmic fractions

  • Only nuclear pCREB1 contributes to transcription

  • Confirm with immunofluorescence microscopy

• ChIP-qPCR or ChIP-seq:

  • Directly measure CREB1 occupancy at target genes

  • Correlate with transcriptional output

• Comprehensive signaling analysis:

  • Assess additional cofactors (TORC, CBP/p300)

  • Evaluate competing factors or repressors

Studies have shown that while phosphorylation at Ser133 generally activates CREB1, the transcriptional outcome depends on multiple factors including cofactor availability and additional modifications .

How can CREB1 antibody pairs be optimized for multiplexed flow cytometry to correlate with functional outcomes?

For multiplexed flow cytometry with CREB1 antibodies:

• Panel design considerations:

  • Select fluorophores with minimal spectral overlap

  • Include phospho-CREB1 (typically PE or Alexa Fluor 488)

  • Include total CREB1 (typically APC or Alexa Fluor 647)

  • Add functional markers (activation, proliferation, apoptosis)

• Sample preparation optimization:

  • Use gentle fixation (0.5-2% paraformaldehyde)

  • Methanol permeabilization for phospho-epitope access

  • Extended antibody incubation (overnight at 4°C)

• Controls for phospho-flow:

  • Biological controls: Stimulated vs. unstimulated

  • Technical controls: FMO (Fluorescence Minus One)

  • Phosphatase-treated negative controls

• Data analysis approaches:

  • Biaxial plots of pCREB1 vs. functional markers

  • Dimensionality reduction (tSNE, UMAP) for population identification

  • Phosphorylation kinetics modeling

Flow cytometry has been successfully used to examine how RRM2 affects cell proliferation induced by CREB1, demonstrating that CREB1 overexpression boosted cell cycle progression while RRM2 knockdown impaired these effects .

What are the most common causes of non-specific binding with CREB1 antibodies and how can they be addressed?

CREB1 belongs to the bZIP family containing both bZIP and KID domains, sharing homology with other transcription factors. Research has demonstrated that antibody specificity can be verified through siRNA knockdown experiments, where silencing CREB1 with siRNA resulted in the down-regulation of RRM2 protein in multiple cell lines .

How can I address epitope masking when detecting phosphorylated CREB1 in complex protein samples?

Epitope masking can occur when protein-protein interactions or conformational changes block antibody access to phosphorylated residues:

• Sample preparation modifications:

  • Denaturing conditions (SDS, heat) for Western blot

  • Extended denaturation time (10 minutes at 95°C)

  • Addition of 8M urea for complete denaturation

  • Methanol fixation for flow cytometry and immunofluorescence

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval (HIER) for FFPE tissues

  • Citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0)

  • Test microwave vs. pressure cooker methods

• Antibody selection strategies:

  • Try multiple antibody clones recognizing different epitopes

  • Consider using antibodies raised against linear epitopes

  • Use phospho-specific antibodies that recognize the flanking sequence

For CREB1 phosphorylation detection, studies have successfully used antibodies against phospho-Ser133 and phospho-Ser129 with proper sample preparation, as evidenced by multiple published protocols .

How do I resolve discrepancies between different antibody-based techniques when measuring CREB1 levels or activity?

When different techniques yield contradictory results:

• Technical comparison approach:

  • Compare antibody performance across methods (WB, IF, IHC, ELISA)

  • Document differences in sample preparation between techniques

  • Test multiple antibody dilutions for each application

• Controls and standards:

  • Include identical positive and negative controls across techniques

  • Use recombinant CREB1 as a reference standard

  • Apply phosphatase treatment to confirm phospho-specificity

• Method-specific considerations:

  • For Western blot: Try different extraction methods and blocking agents

  • For IHC/IF: Test multiple fixation protocols

  • For ELISA: Compare direct vs. sandwich format

  • For flow cytometry: Optimize fixation/permeabilization

• Quantification standardization:

  • Use consistent analysis methods

  • Report relative changes rather than absolute values

  • Always include appropriate loading/housekeeping controls

Research has shown that antibody performance can vary between applications, with some antibodies working well for Western blot but poorly for immunohistochemistry, highlighting the importance of technique-specific validation .

How can I interpret CREB1 phosphorylation patterns in relation to downstream gene expression?

For correlating CREB1 phosphorylation with transcriptional outcomes:

• Integrative analysis approach:

  • Measure pCREB1 by Western blot or ELISA

  • Perform ChIP-seq to identify genomic binding sites

  • Correlate with RNA-seq or qPCR of target genes

  • Calculate correlation coefficients between pCREB1 levels and transcript abundance

• Time-course considerations:

  • Phosphorylation precedes transcriptional changes

  • Typical delay: 30 minutes to several hours

  • Monitor both immediate-early and delayed-response genes

• Contextual factors to consider:

  • Cell type-specific cofactor availability

  • Chromatin accessibility at target loci

  • Competing transcription factors

Research has demonstrated that CREB1 target gene expression significantly discriminates participants who do not acquire HIV-1 post-vaccination in the RV144 trial, with a CREB1 z-score significantly elevated in vaccinated participants who were not infected compared to those who were infected .

What approaches can resolve the functional significance of different CREB1 phosphorylation sites in experimental models?

To determine the functional roles of specific phosphorylation sites:

• Phospho-mimetic/phospho-dead mutants:

  • S133A, S133D, S129A, S129D, S142A, S142D

  • Express in CREB1-knockout cells

  • Measure transcriptional activity and target gene expression

• Phosphorylation site-specific antibodies:

  • Use in ChIP-seq to identify distinct genomic targets

  • Compare cellular localization by immunofluorescence

  • Assess protein-protein interactions by co-IP

• Kinase inhibitor studies:

  • PKA inhibitors for Ser133

  • GSK3β inhibitors for Ser129

  • CaMKII/IV inhibitors for Ser142

  • Monitor site-specific impacts on function

• Mass spectrometry:

  • Quantify relative abundance of different phosphorylation states

  • Identify novel modifications

  • Map combinatorial phosphorylation patterns

Research has shown that phosphorylation of both Ser-133 and Ser-142 in the SCN regulates CREB activity and participates in circadian rhythm generation, while Ser-133 phosphorylation is critical for CREBBP binding and transcriptional activation .

How can CREB1 antibody pairs be effectively utilized in translational research linking preclinical models to clinical outcomes?

For translational applications of CREB1 antibody research:

• Cross-species validation strategy:

  • Confirm antibody cross-reactivity between model organisms and human samples

  • Validate conservation of phosphorylation sites and epitopes

  • Use species-specific positive controls

• Clinical sample considerations:

  • Optimize protocols for FFPE tissues vs. frozen samples

  • Develop standardized IHC scoring systems

  • Correlate with patient metadata and outcomes

• Biomarker development approach:

  • Establish reference ranges in healthy controls

  • Define threshold values for disease states

  • Validate in independent patient cohorts

• Therapeutic response monitoring:

  • Baseline and post-treatment measurements

  • Correlation with treatment efficacy

  • Development of companion diagnostics

Research has demonstrated CREB1's clinical relevance, with studies showing that decreased disease survivals were observed in colorectal cancer patients with high expression levels of CREB1 or RRM2, suggesting potential biomarker utility . Additionally, CREB1 inhibition has shown promise in promoting anti-tumoral immunity in multiple myeloma by limiting HLA-E expression and enhancing NK cell activity .