Phospho-SMAD2 (T220) Antibody

What is Phospho-SMAD2 (T220) Antibody and what epitope does it detect?

Phospho-SMAD2 (T220) Antibody is a specialized immunological reagent that specifically detects SMAD2 protein only when phosphorylated at threonine 220 in the linker region. This antibody recognizes the unique phosphorylated epitope surrounding the T220 residue of human SMAD2, a critical regulatory site in TGF-β signaling . The antibody is highly specific, as it has been designed to detect endogenous levels of phosphorylated SMAD2 without cross-reactivity to the non-phosphorylated form, making it valuable for studying the activation state of this signaling pathway .

How are Phospho-SMAD2 (T220) Antibodies produced and purified?

Phospho-SMAD2 (T220) Antibodies are typically generated through a multi-step immunization and purification process:

• Rabbits are immunized with a KLH (Keyhole Limpet Hemocyanin) conjugated synthetic phosphopeptide corresponding to amino acid residues surrounding T220 of human SMAD2 .

• The resulting antiserum undergoes purification through affinity chromatography using epitope-specific phosphopeptides .

• Non-phospho specific antibodies are removed by additional chromatography using non-phosphorylated peptides .

• Further purification may involve protein A column chromatography followed by peptide affinity purification .

This rigorous production process ensures high specificity for the phosphorylated form of SMAD2 at T220, minimizing cross-reactivity with unphosphorylated SMAD2 or other proteins .

SMAD2 functions as a receptor-regulated SMAD (R-SMAD) that serves as an intracellular signal transducer and transcriptional modulator in the TGF-β signaling pathway . The protein:

• Binds to TRE (TGF-β responsive element) in promoter regions of target genes

• Forms complexes with SMAD4 to activate transcription

• Promotes TGFB1-mediated transcription of differentiation genes

• May act as a tumor suppressor in certain contexts, such as colorectal carcinoma

The phosphorylation of T220 in the linker region is particularly significant as it represents a key regulatory mechanism within the "action turnover switch" model . In this model, T220 phosphorylation by nuclear Cdks creates a docking site for transcriptional co-regulators like Pin1, while potentially enabling subsequent phosphorylation of other linker residues such as Ser-250, which provides binding sites for ubiquitin ligases like Nedd4L that target SMAD2 for proteasomal degradation .

What are the optimal storage conditions for maintaining antibody activity?

To maintain optimal antibody activity and stability, Phospho-SMAD2 (T220) antibodies should be stored following these guidelines:

The antibody is typically supplied in stabilizing buffers that may contain:

• PBS (Phosphate Buffered Saline)

• Glycerol (30-50%)

• Sodium azide (0.01-0.09%)

• BSA (0.5-1%)

These components help maintain antibody stability and prevent microbial contamination. Repeated freeze-thaw cycles should be strictly avoided as they can significantly reduce antibody performance and specificity .

How does SMAD2 T220 phosphorylation integrate into the broader TGF-β signaling network?

SMAD2 T220 phosphorylation represents a critical regulatory node in TGF-β signal transduction that integrates inputs from multiple kinases. Unlike the C-terminal phosphorylation by TGF-β receptor kinases that initiates signaling, T220 phosphorylation occurs primarily in the nucleus by Cyclin-dependent kinases (Cdks) . This creates a multi-layered regulatory system where:

• TGF-β receptor activation leads to C-terminal phosphorylation of SMAD2, causing nuclear translocation

• Once in the nucleus, SMAD2 undergoes T220 phosphorylation by nuclear Cdks

• This phosphorylation creates binding sites for proteins containing WW domains, particularly Pin1

• The interaction with Pin1 modifies SMAD2's conformation, affecting its interaction with transcriptional machinery

• T220 phosphorylation can trigger subsequent phosphorylation of other linker residues (like S250)

• The sequential phosphorylation ultimately targets SMAD2 for ubiquitination and proteasomal degradation

This mechanism represents a sophisticated nuclear "timer" that first enhances and then limits the duration of SMAD2-dependent transcriptional activity, ensuring proper temporal control of TGF-β responsive gene expression.

What experimental considerations are crucial when interpreting results from Phospho-SMAD2 (T220) antibody studies?

When interpreting results from Phospho-SMAD2 (T220) antibody experiments, researchers should consider several critical factors:

Antibody Validation Controls

• Phospho-peptide versus non-phospho-peptide dot blot comparisons are essential to confirm specificity

• Pre-absorption with immunogen peptide should block specific immunoreactivity

• Parallel detection with total SMAD2 antibody is necessary to normalize phosphorylation to total protein levels

Experimental Design Considerations

• Cell type-specific variations in SMAD2 phosphorylation patterns must be acknowledged

• Stimulation conditions (concentration and duration of TGF-β or other agonists) dramatically affect results

• Subcellular localization of phospho-SMAD2 (T220) provides crucial contextual information

• Time-course experiments may be necessary to capture transient phosphorylation events

• Phosphatase inhibitors must be included in sample preparation to prevent ex vivo dephosphorylation

Signal Interpretation

• T220 phosphorylation does not necessarily correlate with canonical C-terminal phosphorylation

• Cross-talk with other signaling pathways (e.g., MAPK, Wnt) can influence T220 phosphorylation independent of TGF-β

• The presence of splice variants (particularly SMAD2 lacking exon 3) must be considered when interpreting band patterns

Researchers should comprehensively document these variables when reporting phospho-SMAD2 (T220) findings to ensure reproducibility and accurate interpretation.

How can researchers optimize immunofluorescence and immunohistochemistry protocols for Phospho-SMAD2 (T220) detection?

Optimizing protocols for Phospho-SMAD2 (T220) detection in IF and IHC applications requires careful attention to several methodological factors:

Fixation and Antigen Retrieval

• For paraffin-embedded tissues, microwave antigen retrieval with 10 mM Tris/EDTA buffer (pH 9.0) has been validated

• For IF on cultured cells, 4% paraformaldehyde fixation followed by permeabilization with 0.1% Triton X-100 is typically effective

• Phospho-epitopes are particularly sensitive to overfixation; optimize fixation time carefully

Antibody Incubation Parameters

• For IHC: dilutions of 1:50-1:300 have been validated

• For IF: dilutions ranging from 1:10-50 or 1:200-1:1000 depending on the specific antibody preparation

• Overnight incubation at 4°C often yields better signal-to-noise ratio than shorter incubations at room temperature

• Secondary antibody selection should match host species (typically anti-rabbit IgG conjugated to appropriate reporter)

Signal Visualization

• For IF: Alexa Fluor 488-conjugated secondary antibodies have been successfully employed

• Counterstaining with phalloidin (for actin) and nuclear stains helps establish subcellular context

• Including positive control samples (e.g., PMA-treated cells) is essential for establishing staining conditions

Validation Approaches

• Peptide competition assays using the phosphorylated immunogen peptide provides critical specificity control

• Parallel staining with antibodies to other SMAD2 phosphorylation sites helps establish phosphorylation patterns

• Treatment with λ-phosphatase can serve as a negative control to confirm phospho-specificity

Careful optimization of these parameters is essential for generating reliable and interpretable results in imaging applications.

What is the functional relationship between different phosphorylation sites in SMAD2?

SMAD2 contains multiple phosphorylation sites that form an interconnected regulatory network:

The "action turnover switch" model suggests a hierarchical relationship where T220 phosphorylation enables subsequent phosphorylation of other linker region residues, particularly S250 . This sequential phosphorylation creates a molecular timer that first promotes SMAD2's transcriptional activity and subsequently targets it for degradation. The interdependence of these phosphorylation events allows for precise temporal control of TGF-β signaling intensity and duration, with T220 playing a pivotal role in this regulatory network.

How can Phospho-SMAD2 (T220) antibodies be used to investigate pathway cross-talk in disease models?

Phospho-SMAD2 (T220) antibodies provide powerful tools for investigating pathway cross-talk in disease models through multiple experimental approaches:

Cancer Research Applications

• Monitor altered T220 phosphorylation patterns in tumor samples compared to matched normal tissues using IHC

• Investigate correlation between T220 phosphorylation status and cancer stage/prognosis

• Examine how oncogenic signaling pathways (RAS/MAPK, PI3K/AKT) impact T220 phosphorylation patterns

• Evaluate therapeutic response by monitoring T220 phosphorylation after treatment with pathway inhibitors

Fibrosis Model Applications

• Track T220 phosphorylation in progressive fibrosis using the antibody in Western blot and IHC applications

• Correlate T220 phosphorylation with expression of fibrotic markers and disease severity

• Investigate how anti-fibrotic interventions affect T220 phosphorylation dynamics

Methodological Approaches

• Co-immunoprecipitation with Phospho-SMAD2 (T220) antibody can identify novel interaction partners

• Multiplex immunofluorescence combining Phospho-SMAD2 (T220) with markers of other pathways

• Pharmacological manipulation studies using pathway-specific inhibitors followed by assessment of T220 phosphorylation

• siRNA/CRISPR approaches targeting specific pathway components to assess their impact on T220 phosphorylation

Technical Considerations

• Use of multiple antibodies targeting different phosphorylation sites provides a comprehensive view of SMAD2 regulation

• Time-course experiments are crucial for capturing dynamic changes in phosphorylation status

• Cell type-specific effects must be considered when interpreting results

Through these approaches, Phospho-SMAD2 (T220) antibodies can reveal novel mechanistic insights into how TGF-β signaling intersects with other pathways in disease pathogenesis, potentially identifying new therapeutic targets.