Phospho-ERF (T526) Antibody

What is Phospho-ERF (T526) Antibody and what does it specifically detect?

Phospho-ERF (T526) Antibody is a research tool designed to detect the ETS domain-containing transcription factor ERF (Ets2 repressor factor) only when phosphorylated at threonine 526. This antibody is critical for studying post-translational modifications that regulate ERF activity. The antibody recognizes the specific phosphorylated epitope surrounding Thr526, typically derived from a synthetic phosphopeptide immunogen from human ERF encompassing amino acids 492-541 .

ERF functions as a transcriptional repressor within the ETS family, playing important roles in regulating both proliferation and differentiation. The phosphorylation status at Thr526 is particularly significant for modulating ERF's transcriptional repressor activity and its involvement in cellular processes .

What are the validated applications for Phospho-ERF (T526) Antibody?

Based on extensive validation data, Phospho-ERF (T526) Antibody has been validated for the following applications:

The antibody has been specifically tested in these applications to ensure reliable and reproducible results across various experimental conditions .

How does phosphorylation at T526 affect ERF function?

Phosphorylation of ERF at threonine 526 plays a critical role in regulating its biological activity. ERF is a ubiquitously expressed transcription factor that exhibits strong transcriptional repressor activity. Its phosphorylation at T526 has been specifically linked to:

• Regulation of ERF's nuclear export and subcellular localization

• Modulation of its DNA binding affinity

• Control of its transcriptional repressor activity

• Involvement in cellular processes including proliferation, differentiation, and apoptosis

The phosphorylation state of ERF at T526 is regulated by MAPK signaling pathways, with this post-translational modification serving as a key mechanism for controlling ERF's involvement in the transcriptional regulation of genes activated during cell cycle progression, particularly during entry into G1 phase .

What are the optimal storage conditions for Phospho-ERF (T526) Antibody?

For maximum antibody performance and stability, follow these storage recommendations:

• Long-term storage: Store at -20°C for up to one year

• Short-term/frequent use: Store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles to maintain antibody activity

• The antibody is typically supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide to enhance stability

Proper aliquoting upon first thaw is recommended to minimize freeze-thaw cycles. When stored properly, the antibody should maintain its specificity and sensitivity for the validated shelf life period .

What controls should be included when using Phospho-ERF (T526) Antibody?

Including appropriate controls is essential for accurate interpretation of results with Phospho-ERF (T526) Antibody:

Positive controls:

• Cell lysates known to express phosphorylated ERF (T526), such as growth factor-stimulated cells

• Tissues with documented ERF phosphorylation (e.g., human lung carcinoma tissue has been validated)

Negative controls:

• Samples treated with phosphatase to remove phosphorylation

• Blocking with the specific phosphopeptide immunogen (blocking peptide testing has been demonstrated in validation images)

Technical controls:

• Isotype control antibody (rabbit IgG) to assess non-specific binding

• Secondary antibody-only control to evaluate background

• Omission of primary antibody to determine secondary antibody specificity

How can specificity of Phospho-ERF (T526) Antibody be verified?

Verifying antibody specificity is critical for ensuring reliable experimental results. For Phospho-ERF (T526) Antibody, consider these approaches:

• Peptide competition assay: Pre-incubate the antibody with the phospho-peptide immunogen before application to your sample. Specific signals should be blocked or significantly reduced, as demonstrated in validation images for this antibody .

• Phosphatase treatment: Treat half of your sample with lambda phosphatase to remove phosphate groups. The signal should disappear or diminish in the phosphatase-treated sample.

• Activation/inhibition experiments: Treat cells with activators (e.g., growth factors) or inhibitors (e.g., MAPK pathway inhibitors) known to affect ERF phosphorylation and confirm expected changes in signal intensity.

• Immunoprecipitation followed by mass spectrometry: For advanced validation, immunoprecipitate with the antibody and confirm the identity and phosphorylation status of the captured protein .

What experimental designs are suitable for studying ERF phosphorylation dynamics?

When designing experiments to study ERF phosphorylation dynamics, consider these validated approaches:

• Time-course experiments: Treat cells with growth factors or other stimuli and collect samples at different time points to track phosphorylation changes.

• Dose-response studies: Expose cells to varying concentrations of stimuli or inhibitors to determine threshold effects on ERF phosphorylation.

• Quasi-experimental designs: When clinical samples are involved, carefully design studies with appropriate controls, especially when randomization is not possible. Prepost designs with nonequivalent control groups, interrupted time series, and stepped-wedge designs are recommended for balancing internal and external validity .

• Genetic manipulation approaches: Use gene editing (CRISPR/Cas9) to create phospho-mimetic (T526E) or phospho-deficient (T526A) ERF mutants to study functional consequences of phosphorylation.

• Subcellular fractionation: Combine with western blotting to track changes in the subcellular localization of phosphorylated ERF under different conditions .

How should phosphorylation patterns be interpreted across different tissue or cell types?

Interpretation of ERF phosphorylation patterns requires careful consideration of biological context:

• Basal phosphorylation levels: Different tissues naturally exhibit varying basal levels of ERF phosphorylation based on their proliferative status and signaling activity. Document these baseline differences before making comparisons.

• Cell type-specific signaling: The upstream kinases responsible for ERF T526 phosphorylation may vary between cell types. Consider characterizing the relevant signaling pathways in your specific model.

• Functional consequences: The same phosphorylation event may have different consequences in different cellular contexts. Complement phosphorylation analysis with functional assays (e.g., transcriptional reporter assays).

• Quantitative analysis: Use appropriate statistical methods when comparing phosphorylation levels between samples. Consider normalizing phospho-ERF signal to total ERF levels to account for expression differences .

• Correlation with physiological state: In tissue samples, correlate phosphorylation patterns with histopathological features, clinical parameters, or disease progression to establish physiological relevance .

Why might Western blot using Phospho-ERF (T526) Antibody show no signal or multiple bands?

No signal issues:

• Insufficient phosphorylation of ERF in samples (try using positive controls like growth factor-stimulated cells)

• Inadequate antibody concentration (optimize dilution; recommended range is 1:500-1:2000 for WB)

• Loss of phosphorylation during sample preparation (include phosphatase inhibitors)

• Inefficient transfer of high molecular weight proteins (optimize transfer conditions)

• Degraded antibody (check storage conditions and expiration date)

Multiple bands issues:

• Cross-reactivity with other phosphorylated proteins (verify with blocking peptide)

• ERF isoforms or post-translationally modified variants

• Proteolytic degradation of ERF (ensure complete protease inhibition)

• Non-specific binding (optimize blocking conditions and antibody dilution)

• Splice variants of ERF (ERF has a molecular weight of approximately 58-59 kDa)

How can immunohistochemistry results be optimized when using Phospho-ERF (T526) Antibody?

For optimal immunohistochemistry results:

• Antigen retrieval optimization:

  • Test both heat-induced epitope retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Compare different retrieval times (10-30 minutes) to determine optimal conditions

  • Phospho-epitopes are often sensitive to fixation; freshly prepared sections may yield better results

• Antibody dilution:

  • Start with the recommended range (1:100-1:300)

  • Prepare a dilution series to determine optimal concentration for your specific tissue

  • Incubate overnight at 4°C for maximum sensitivity

• Background reduction:

  • Ensure thorough blocking (3-5% BSA or normal serum from the species of secondary antibody)

  • Include 0.1-0.3% Triton X-100 for better antibody penetration

  • Consider adding 0.05% Tween-20 to wash buffers

  • Use validated detection systems appropriate for phospho-epitopes

How can Phospho-ERF (T526) Antibody be used to study MAPK signaling pathways?

Phospho-ERF (T526) Antibody provides a valuable downstream readout for MAPK pathway activity:

• Pathway activation studies:

  • Monitor ERF phosphorylation after stimulation with growth factors known to activate MAPK pathways

  • Use in conjunction with phospho-specific antibodies against MEK and ERK to build a complete pathway profile

  • Compare kinetics of ERF phosphorylation with those of upstream components

• Inhibitor screening:

  • Evaluate the effects of different MAPK pathway inhibitors on ERF phosphorylation

  • Use as a functional readout in drug discovery efforts targeting MAPK signaling

  • Establish dose-response relationships between inhibitor concentration and ERF phosphorylation levels

• Signaling crosstalk:

  • Investigate how other signaling pathways influence ERF phosphorylation at T526

  • Use pathway-specific activators and inhibitors to map signaling network interactions

  • Employ phospho-proteomics approaches to place ERF phosphorylation in broader signaling context

What are the relationships between ERF phosphorylation and cell cycle regulation?

ERF phosphorylation at T526 has important implications for cell cycle control:

• Cell cycle phase correlation:

  • Track ERF phosphorylation throughout the cell cycle using synchronized cell populations

  • Correlate with expression of cyclins and CDK activities

  • ERF phosphorylation has been specifically linked to transcriptional regulation during G1 phase entry

• Growth arrest studies:

  • Examine changes in ERF phosphorylation during serum starvation and restimulation

  • Investigate the relationship between ERF phosphorylation status and contact inhibition

  • Analyze how ERF phosphorylation affects expression of cell cycle regulators

• Proliferation and differentiation:

  • ERF exhibits strong transcriptional repressor activity that suppresses Ets-induced transformation

  • Study how changes in phosphorylation at T526 influence the transition between proliferative and differentiated states

  • Investigate potential roles in terminal differentiation of specific cell lineages